B7-related nucleic acids and polypeptides useful for immunomodulation

ABSTRACT

The present invention provides nucleic acids encoding B7-related factors that modulate the activation of immune or inflammatory response cells, such as T-cells. Also provided are expression vectors and fusion constructs comprising nucleic acids encoding B7-related polypeptides, including BSL1, BSL2, and BSL3. The present invention further provides isolated B7-related polypeptides, isolated fusion proteins comprising B7-related polypeptides, and antibodies that are specifically reactive with B7-related polypeptides, or portions thereof. In addition, the present invention provides assays utilizing B7-related nucleic acids, polypeptides, or peptides. The present invention further provides compositions of B7-related nucleic acids, polypeptides, fusion proteins, or antibodies that are useful for the immunomodulation of a human or animal subject.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.09/875,338, filed Jun. 6, 2001, which is a continuation-in-part of U.S.Application Ser. No. 60/272,107, filed Feb. 28, 2001, and U.S.Application Ser. No. 60/209,811, filed Jun. 6, 2000, which are allhereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to isolated nucleic acids encodingB7-related polypeptides, including BSL1, BSL2, and BSL3, which modulatecells that are important for immune and inflammatory responses, such asT-cells. Also related are expression vectors and fusion constructscomprising nucleic acids encoding B7-related polypeptides. The presentinvention further relates to isolated B7-related polypeptides, isolatedfusion proteins comprising B7-related polypeptides, and antibodies thatare specifically reactive with B7-related polypeptides, or portionsthereof. In addition, the present invention relates to methods ofisolating and identifying the corresponding counter-receptor(s) of theB7-related polypeptides, utilizing B7-related polypeptides, or fusionproteins. Also related are methods of immunomodulation of a subject bythe administration of compositions of the B7-related polypeptides,fusion proteins, cognate antibodies, or portions or derivatives thereof.The present invention further relates to methods of immunomodulation ofanimal or human subjects by the administration of compositions ofgenetically engineered vectors or host cells comprising the B7-relatedpolypeptide expression cassettes as disclosed herein.

BACKGROUND OF THE INVENTION

The primary response of T-cells, involving T-cell activation, expansion,and differentiation is essential for the initiation of an immuneresponse to a foreign antigen. The activation of T-cells by antigenpresenting cells (APCs) requires at least two separate signals (K. E.Hellstrom et al. (1996) Cancer Chemother. Pharmacol. 38:S40-1; N. K.Damle et al. (1992) J. Immunol. 148:1985-92; J. M. Green et al. (1994)Eur. J. Immunol. 24:265-72; E. C. Guinan et al. (1994) Blood 84:3261-82;J. W. Hodge et al. (1995) Cancer Res. 55:3598-603). The first signalcauses T-cell entry into the cell cycle, and is mediated by foreignantigens presented by the major histocompatibility complex (MHC). Thesecond signal, termed costimulation, causes cytokine production andT-cell proliferation, but is neither antigen-specific, nor MHCrestricted (R. H. Schwartz (1990) Science 248:1349-1356).

Costimulation is believed to be mediated by one or more distinct cellssurface molecules expressed by APCs (M. K. Jenkins et al. (1988) J.Immunol. 140:3324-3330; P. S. Linsley et al. (1991) J. Exp. Med.173:721-730; C. D. Gimmi, et al. (1991) Proc. Natl. Acad. Sci. USA88:6575-6579; J. W. Young et al. (1992) J. Clin. Invest. 90:229-237; L.Koulova et al. (1991) J. Exp. Med. 173:759-762; H. Reiser et al. (1992)Proc. Natl. Acad. Sci. USA 89:271-275; G. A. van-Seventer et al. (1990)J. Immunol. 144:4579-4586; J. M. LaSalle et al. (1991) J. Immunol.147:774-80; M. I. Dustin et al. (1989) J. Exp. Med. 169:503; R. J.Armitage et al. (1992) Nature 357:80-82; Y. Liu et al. (1992) J. Exp.Med. 175:437-445). Considerable evidence suggests that B7, an APCcell-surface protein, is one such costimulatory molecule (P. S. Linsleyet al. (1991) J. Exp. Med. 173:721-730; C. D. Gimmi et al. (1991) Proc.Natl. Acad. Sci. USA 88:6575-6579; L. Koulova et al. (1991) J. Exp. Med.173:759-762; H. Reiser et al. (1992) Proc. Natl. Acad. Sci. USA 89,271-275; P. S. Linsley et al. (1990) Proc. Natl. Acad. Sci. USA87:5031-5035; G. J. Freeman et al. (1991) J. Exp. Med. 174:625-631).

B7 has been shown to bind to two counter-receptors (ligands) expressedon T-cells, termed CD28 and CTLA-4. B7 binding to CD28 induces T-cellsto proliferate and secrete IL-2 (P. S. Linsley et al. (1991) J. Exp.Med. 173, 721-730; C. D. Gimmi et al. (1991) Proc. Natl. Acad. Sci. USA88:6575-6579; C. B. Thompson et al. (1989) Proc. Natl. Acad. Sci. USA86:1333-1337; C. H. June et al. (1990) Immunol. Today 11:211-6; F. A.Harding et al. (1992) Nature 356:607-609), allowing full T-cellactivation. Conversely, B7 binding to CTLA-4 mediates T-celldown-regulation. The importance of the B7:CD28/CTLA-4 costimulatorypathway has been demonstrated in vitro and in several in vivo modelsystems. Blockade of this pathway results in the development of antigenspecific tolerance in murine and humans systems (F. A. Harding et al.(1992) Nature 356:607-609; D. J. Lenschow et al. (1992) Science257:789-792; L. A. Turka et al. (1992) Proc. Natl. Acad. Sci. USA89:11102-11105; C. D. Gimmi et al. (1993) Proc. Natl. Acad. Sci. USA90:6586-6590). Conversely, the ectopic expression of B7 in B7-negativemurine tumor cells induces T-cell mediated specific immunity accompaniedby tumor rejection and long lasting protection to tumor challenge (L.Chen et al. (1992) Cell 71:1093-1102; S. E. Townsend et al. (1993)Science 259:368-370; S. Baskar et al. (1993) Proc. Natl. Acad. Sci. USA90:5687-5690). Therefore, manipulation of the B7:CD28/CTLA-4 pathwayoffers great potential to stimulate or suppress immune responses inhumans.

In addition to the previously characterized B7 molecule (referred tohereafter as B7-1) B7-1-like molecules have been identified (see, e.g.,M. Azuma et al. (1993) Nature 366:76-79; C. Chen et al. (1994) J.Immunol. 152:4929-36; R. H. Reeves et al. (1997) Mamm. Genome 8:581-582;K. Ishikawa et al. (1998) DNA Res. 5:169-176; U.S. Pat. No. 5,942,607issued Aug. 24, 1999 to Freeman et al.). In particular, PD-L1 and PD-L2have been identified as inhibitors of T-cell activation (G. J. Freemanet al. (2000) J. Exp. Med. 192:1027-1034; Y. Latchman et al., (2001)Nature Immunology 2:261-268), whereas B7-H1, B7-H3, and B7-DC have beendescribed as co-stimulators of T-cell proliferation (H. Dong et al.(1999) Nature Medicine 5:1365-1369; A. I. Chapoval (2001) NatureImmunology 2:269-274; Tseng et al. (2001) J. Exp. Med. 193(7):839-45).

Thus, there is a growing family of factors related to B7-1, whichmodulate T-cell activation (reviewed by J. Henry et al. (1999) Immunol.Today 20:285-288). The identification, isolation, and characterizationof B7-related factors are therefore important goals for the furtherunderstanding of T-cell activation and function in both normal anddisease states in animals, particularly humans. Accordingly, the presentinvention discloses the discovery and characterization of threeB7-related factors, termed BSL1, BSL2, and BSL3. Also disclosed arevarious assays and treatments utilizing the BSL1, BSL2, and BSL3factors.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide isolated nucleicacids encoding B7-related polypeptides that modulate inflammatory andimmune responses, including T-cell activation (i.e., lymphokineproduction and/or T-cell proliferation). B7-related polypeptides withinthe scope of the invention include counter-receptors on the surface ofAPCs capable of binding CD28/CTLA-4 and/or CD28-/CTLA-4-relatedligand(s). Specifically, B7-related polypeptides include the BSL1, BSL2,and BSL3 polypeptides, and soluble fragments or derivatives thereof.More specifically, a B7-related nucleic acid is: i) a nucleic acidmolecule comprising at least a fragment of a nucleotide sequenceencoding a BSL1 (e.g., SEQ ID NO:2), BSL2 (e.g., SEQ ID NO:7, 11, or13), or BSL3 (e.g., SEQ ID NO:15) polypeptide; ii) a nucleic acidmolecule comprising a nucleotide sequence encoding a polypeptide thatshares moderate to substantial sequence homology with a BSL1 (e.g., SEQID NO:2), BSL2 (e.g., SEQ ID NO:7, 11, or 13), or BSL3 (e.g., SEQ IDNO:15) polypeptide; iii) a nucleic acid molecule capable of hybridizingto the BSL1 (e.g., SEQ ID NO:1 or 3), BSL2 (e.g., SEQ ID NO:6, 10, 12,or 131), or BSL3 (e.g., SEQ ID NO:14) nucleotide sequences, or fragmentsthereof, under appropriate conditions (e.g., moderate or high stringencyhybridization conditions); iv) a nucleic acid molecule which differsfrom the nucleotide sequence of BSL1 (e.g., SEQ ID NO:1 or 3), BSL2(e.g., SEQ ID NO:6, 10, 12, or 131), or BSL3 (e.g., SEQ ID NO:14) due todegeneracy in the genetic code, or recombinant or syntheticmodifications; or v) a nucleic acid molecule that shares at leastsubstantial homology with the nucleic acid sequence set forth in SEQ IDNO:1, 3, 6, 10, 12, 14, or 131.

In addition, nucleic acid probes or primers comprising B7-relatedsequences are encompassed by the present invention. Such probes andprimers are useful, for example, for assaying a biological sample forthe presence of APCs expressing the BSL1, BSL2, and BSL3 factors.

It is another object of the present invention to provide vectors (e.g.,expression vectors) and fusion constructs comprising nucleic acidsencoding B7-related polypeptides. Expression vectors direct thesynthesis of the corresponding polypeptides or peptides in a variety ofhosts, particularly-eukaryotic cells, such as mammalian and insect cellculture, and prokaryotic cells, such as Escherichia coli. Expressionvectors within the scope of the invention comprise a nucleic acidsequence encoding at least one B7-related polypeptide as describedherein, and a promoter operatively linked to the nucleic acid sequence.In one embodiment, the expression vector comprises a DNA sequenceencoding the extracellular domain of BSL1 (e.g., SEQ ID NO:2), BSL2(e.g., SEQ ID NO:7, 11, or 13), or BSL3 (e.g., SEQ ID NO:15) fused to aDNA sequence encoding the Fc region human immunoglobulin G1 (IgG1). Suchexpression vectors can be used to transform or transfect host cells tothereby produce polypeptides or peptides, including fusion proteins orpeptides encoded by nucleic acid molecules as described herein.

It is yet another object of the present invention to provide isolatedB7-related polypeptides, including the BSL1, BSL2, and BSL3polypeptides, or portions or derivatives thereof. Preferred B7-relatedpolypeptides comprise the amino acid sequences of the BSL1 (e.g., SEQ IDNO:2), BSL2 (e.g., SEQ ID NO:7, 11, or 13), or BSL3 (e.g., SEQ ID NO:15)polypeptides, or portions thereof. Such polypeptides comprise at least aportion of the mature forms of the BSL1, BSL2, and BSL3 polypeptides,and preferably comprise soluble forms of these polypeptides. Alsoencompassed by the present invention are polypeptides that sharemoderate to substantial homology with the amino acid sequence set forthin SEQ ID NO:2, 7, 11, 13, or 15, which are naturally occurring isoformsof the BSL1, BSL2, or BSL3 polypeptides, or modified recombinantpolypeptides.

It is still another object of the present invention to provide isolatedfusion proteins comprising the B7-related polypeptides, or portions orderivatives thereof, as disclosed herein. In one aspect, the fusionprotein comprises an extracellular domain portion of a B7-relatedpolypeptide fused to another polypeptide that alters the solubility,purification, binding affinity, and/or valency of the B7-relatedpolypeptide. Preferably, a DNA molecule encoding an extracellular domainportion of the BSL1, BSL2, or BSL3 polypeptides can be joined to DNAencoding the Fc region of human IgG1 to form DNA fusion products thatencode the BSL1-Ig (e.g., SEQ ID NO:5), BSL2-Ig (e.g., SEQ ID NO:9, 133,or 135), or BSL3-Ig (e.g., SEQ ID NO:17) fusion proteins.

It is a further object of the present invention to provide methods ofisolating and identifying the corresponding counter-receptor(s) of theB7-related polypeptides, utilizing the isolated B7-related polypeptides,fusion proteins, or cognate antibodies disclosed herein. In oneembodiment, isolated BSL1, BSL2, or BSL3 polypeptides, or portionsthereof, can be incubated with protein extracts obtained from immune orinflammatory response cells, such as T-cells, to form a BSUreceptorcomplex, and then incubated with anti-BSL antibodies to isolate theBSUreceptor complex. Alternatively, a fusion protein comprising theBSL1, BSL2, or BSL3 polypeptide can be incubated with protein extractsobtained from immune or inflammatory response cells, such as T-cells,and then incubated with antibodies that specifically react with thefusion protein. Receptors that bind to the B7-related polypeptides wouldbe expected to have significant immunomodulatory activity.

It is another object of the present invention to provide diagnosticmethods and kits utilizing the B7-related factors of the presentinvention, including nucleic acids, polypeptides, antibodies, orfunctional fragments thereof. Such factors can be used, for example, indiagnostic methods and kits for measuring expression levels ofB7-related factors, and to screen for various B7-related diseases. Inaddition, the B7-related nucleic acids described herein can be used toidentify chromosomal abnormalities affecting BSL1, BSL2, or BSL3, and toidentify allelic variants or mutations of BSL1, BSL2, or BSL3 in anindividual or population.

It is yet another object of the present invention to provide isolatedantibodies, including monoclonal and polyclonal antibodies, that arespecifically reactive with the B7-related polypeptides, fusion proteins,or portions or derivatives thereof, as disclosed herein. Preferably,monoclonal antibodies are prepared to be specifically-reactive with theBSL1 (e.g., SEQ ID NO:2), BSL2 (e.g., SEQ ID NO:7, 11, or 13), or BSL3(e.g., SEQ ID NO:15) polypeptides, or portions or derivatives thereof.

It is another object of the present invention to provide methods ofimmunomodulation of a human or animal subject by the administration ofcompositions of the B7-related polypeptides, fusion proteins, orportions or derivatives thereof, as disclosed herein. Such compositionswould be expected to up-regulate or down-regulate the activities ofimmune or inflammatory response cells (e.g., T-cells). For example,B7-related polypeptides in a composition may interact with CD28 andthereby up-regulate immune cell activity. Alternatively, B7-relatedpolypeptides in a composition may interact with CTLA-4 and therebydown-regulate immune cell activity. In one embodiment, compositions ofBSL1-Ig, BSL2-Ig, and BSL3-Ig, fusion proteins are administered, e.g.via injection, to a subject to provide systemic immunosupression orimmunostimulation. In a specific embodiment, BSL2-Ig (e.g., SEQ ID NO:9)fusion proteins can be used to inhibit T-cell proliferation, and therebytreat conditions associated with aberrant or increased T-cellproliferation. The protein or fusion protein compositions of theinvention can be administered alone, or in combination with one or moreimmunomodulatory molecules. For example, BSL2-Ig fusion proteins (e.g.,SEQ ID NO:9) can be administered in combination with antibodies againsta BSL2 polypeptide (e.g., SEQ ID NO:7) to inhibit T-cell proliferation.

It is still another object of the present invention to provide methodsof immunomodulation of a human or animal subject by the administrationof compositions of antibodies that are specifically reactive with theB7-related polypeptides, fusion proteins, or portions or derivativesthereof, as disclosed herein. Such compositions can be expected to blockthe co-stimulatory activities of the B7-related polypeptides, and todown-regulate immune or inflammatory response cells (e.g., T-cells),accordingly. In one embodiment, compositions of monoclonal antibodiesthat are specifically reactive with the BSL1 (e.g., SEQ ID NO:2), BSL2(e.g., SEQ ID NO:7, 11, or 13), or BSL3 (e.g., SEQ ID NO:15)polypeptides, or fragments thereof, are administered, e.g., viainjection, to a subject to provide immunosupression or inducedtolerance. In a specific embodiment, monoclonal antibodies against aBSL2 polypeptide (e.g., SEQ ID NO:7) can be used to inhibit T-cellproliferation, and thereby treat conditions associated with aberrant orincreased T-cell proliferation. Antibody compositions can beadministered alone, or in combination with one or more immunomodulatorymolecules. For example, antibodies against a BSL2 polypeptide (e.g., SEQID NO:7) can be administered in combination with a BSL2-Ig fusionprotein (e.g., SEQ ID NO:9) to inhibit T-cell proliferation. The methodsof inducing tolerance described herein can be used prophylactically forpreventing immune responses such as transplantation rejection (solidorgan and bone marrow) and graft versus host disease, especially inautologous bone marrow transplantation. Such methods can also be usefultherapeutically, in the treatment of autoimmune diseases,transplantation rejection, and established graft versus host disease ina subject.

It is a further object of the present invention to provide methods ofthe immunomodulation of a human or animal subject by the administrationof compositions of genetically engineered vectors or cells comprisingthe B7-related polypeptide expression cassettes as disclosed herein. Ina preferred embodiment, the cells are antigen presenting cells, such asa macrophages, which are transfected or transduced to allow expressionof one or more of the B7-related polypeptides, including the BSL1 (e.g.,SEQ ID NO:2), BSL2 (e.g., SEQ ID NO:7, 11, or 13), or BSL3 (e.g., SEQ IDNO:15) polypeptides, fusion proteins, or fragments or derivativesthereof, and then introduced e.g., via transplantation, into therecipient. Consistent with the present invention, the genes encoding theBSL1, BSL2, or BSL3 polypeptides or fusion proteins can be transfectedor transduced alone, or in combination with genes encoding otherimmunomodulatory molecules.

Additional objects and advantages afforded by the present invention willbe apparent from the detailed description and exemplification hereinbelow.

BRIEF DESCRIPTION OF THE FIGURES

The appended drawings of the figures are presented to further describethe invention and to assist in its understanding through clarificationof its various aspects. In the figures of the present invention, thenucleotide and amino acid sequences are represented by their one-letterabbreviations.

FIGS. 1A-1C illustrate the nucleotide and predicted amino acid sequenceof BSL1. FIG. 1A shows the nucleotide sequence of BSL1 (SEQ ID NO:1)determined from the full-length clone isolated from a cDNA libraryprepared from human microvascular endothelial cells treated withTNF-alpha; nucleotides 1-92 contain the 5′-untranslated region;nucleotides 93-95 contain the translation initiation signal (ATG);nucleotides 93-962 contain the protein coding region; nucleotides963-965 contain the translation termination signal (TAA); nucleotides963-1576 contain the 3′-untranslated region; and nucleotides 1577-1605contain the poly(A)⁺ RNA tail. FIG. 1B shows the predicted amino acidsequence of BSL1 (SEQ ID NO:2); amino acids 1-240 contain the predictedextracellular domain (ECD). FIG. 1C shows the nucleotide sequence ofBSL1 (SEQ ID NO:3) determined from the full-length clone isolated from acDNA library prepared from GM-CSF/IL-4 differentiated human monocytecells; nucleotides 1-92 contain the 5′-untranslated region; nucleotides93-95 contain the translation initiation signal (ATG); nucleotides93-962 contain the protein coding sequence; nucleotides 963-965 containthe translation stop signal (TAA); and nucleotides 963-3621 contain the3′-untranslated region (unique sequence is shown in bold).

FIGS. 2A-2B illustrate the nucleotide and predicted amino acid sequenceof the BSL1-Ig fusion construct. FIG. 2A shows the nucleotide sequenceof the BSL1-Ig fusion construct (SEQ ID NO:4); nucleotides 1-72 encodethe predicted CD5 signal sequence; nucleotides 73-78 contain the SpeIrestriction site; nucleotides 78-729 encode the predicted BSL1 ECD;nucleotides 730-1440 encode the Fc portion of human IgG1; andnucleotides 1441-1443 contain the translation stop signal (TGA). FIG. 2Bshows the BSL1-Ig predicted amino acid sequence (SEQ ID NO:5).

FIGS. 3A-3H illustrate the nucleotide and predicted amino sequences ofthe BSL2 clones. FIG. 3A shows the nucleotide sequence of theBSL2-4616811 clone (SEQ ID NO:6); nucleotides 1-12 include vectorsequence; nucleotides 121-123 contain the translation initiation signal(ATG); between nucleotides 204-205 is the predicted signal peptidecleavage site; nucleotides 1516-1587 encode the predicted transmembranedomain; nucleotides 1723-1725 contain the translation termination signal(TGA). It is noted that BSL2-4616811 is also called BSL2vcvc for thepurposes of this invention. FIG. 3B shows the predicted amino acidsequence of the BSL2-4616811 clone (SEQ ID NO:7); amino acids 1-465contain the predicted ECD. The sequence of the mature BSL2-4616811polypeptide begins at amino acid 29. FIG. 3C shows the nucleotidesequence of the BSL2-L165-21 clone (SEQ ID NO:10); nucleotides 1-3contain the translation initiation signal (ATG); between nucleotides84-85 is the predicted signal peptide cleavage site; nucleotides 742-813encode the predicted transmembrane domain; nucleotides 949-951 containthe translation termination signal (TGA). It is noted that BSL2-L165-21is also called BSL2v2c2 for the purposes of this invention. FIG. 3Dshows the predicted amino acid sequence of the BSL2-L165-21 clone (SEQID NO:11); amino acids 1-247 contain the predicted ECD. FIG. 3E showsthe nucleotide sequence of the BSL2-L165-35b clone (SEQ ID NO:12);nucleotides 1-3 contain the translation initiation signal (ATG); betweennucleotides 84-85 is the predicted signal peptide cleavage site;nucleotides 742-813 encode the predicted transmembrane domain;nucleotides 949-951 contain the translation termination signal (TGA).The sequence encoding the mature BSL2-L165-35b polypeptide begins atnucleotide 85. It is noted that BSL2-L165-35b is also called BSL2v1c2for the purposes of this invention. FIG. 3F shows the predicted aminoacid sequence of the BSL2-L165-35b clone (SEQ ID NO:13); amino acids1-247 contain the predicted ECD. The sequence encoding the matureBSL2-L165-35b polypeptide begins at amino acid 29. FIG. 3G shows thecoding sequence of BSL2-4616811 (BSL2vcvc; SEQ ID NO:131). The sequenceencoding the mature form of the BSL2-4616811 polypeptide begins atnucleotide 85; nucleotides 1-3 contain the translation initiation signal(ATG); and nucleotides 1396-1467 encode the predicted transmembranedomain. FIG. 3H shows the exons and alternative splicing diagram for theBSL2 clones, including BSL2-4616811 (BSL2vcvc), BSL2-L165-21 (BSL2v2c2),and BSL2-L165-35b (BSL2v1c2). In the diagram, the exons are not drawn toscale, and the first 66 nucleotides of the BSL2-4616811 clone are notmapped to the genomic sequence.

FIGS. 4A-4F illustrate the nucleotide and predicted amino sequences ofthe BSL2-4616811-Ig (BSL2vcvc-Ig), BSL2-L165-35b-Ig (BSL2v1c2-Ig), andBSL2-L165-21-Ig (BSL2v2c2-Ig) fusion constructs. FIG. 4A shows thenucleotide sequence of the BSL2-4616811-Ig clone (SEQ ID NO:8);nucleotides 1-3 contain the translation initiation signal (ATG);nucleotides 1-1394 encode the BSL2-4616811 ECD; nucleotide 1395 is asilent mutation introduced to facilitate construction of the fusionprotein; nucleotides 1396-2094 encode the Fc portion of human IgG1;nucleotides 2095-2097 contain the translation termination signal (TGA).FIG. 4B shows the predicted amino acid sequence of the BSL2-4616811-Igfusion protein (SEQ ID NO:9); amino acids 1-465 of contain theBSL2-4616811 ECD; amino acids 85-465 contain the mature BSL2-4616811ECD; amino acids 466-498 contain the Fc domain of human IgG. FIG. 4Cshows the nucleotide sequence of the BSL2-L165-35b-Ig clone (SEQ IDNO:132); nucleotides 1-3 contain the translation initiation signal(ATG); nucleotides 1-84 encode the predicted signal peptide sequence;nucleotides 85-738 encode the mature ECD; nucleotides 739-744 contain arestriction site introduced by PCR to facilitate construction of thefusion; and nucleotides 745-1440 encode the human Ig portion of thefusion construct. FIG. 4D shows the predicted amino acid sequence of theBSL2-L165-35b-Ig fusion protein (SEQ ID NO:133); amino acids 1-28contain the predicted signal peptide sequence; amino acids 29-226contain the mature ECD; amino acids 227-228 correspond to therestriction site introduced by PCR; amino acids 229-480 contain thehuman Ig portion of the fusion. FIG. 4E shows the nucleotide sequence ofthe BSL2-L165-21-Ig clone (SEQ ID NO:134); nucleotides 1-3 contain thetranslation initiation signal (ATG); nucleotides 1-84 encode thepredicted signal peptide sequence; nucleotides 85-738 encode thepredicted mature ECD; nucleotides 739-744 contain an EcoRI siteintroduced by PCR to facilitate construction of the fusion; nucleotides745-1440 encode the human Ig portion of the fusion construct. FIG. 4Fshows the predicted amino acid sequence of the BSL2-L165-21-Ig fusionprotein (SEQ ID NO:135); amino acid 1 is the initiating methionine;amino acids 1-28 contain the predicted signal peptide sequence; aminoacids 29-246 contain the predicted mature ECD; amino acids 247-248correspond to the EcoRI restriction site introduced by PCR; amino acids249-480 contain the human Ig portion of the fusion protein.

FIGS. 5A-5B illustrate the nucleotide and predicted amino acid sequenceof BSL3. FIG. 5A shows the nucleotide sequence of BSL3 (SEQ ID NO:14):nucleotides 1-326 contain 5′ untranslated region; nucleotides 327-329contain the translation initiation signal (ATG); nucleotides 981-1055encode a predicted transmembrane domain; nucleotides 1146-1148 containthe translation termination signal (TGA) FIG. 5B shows the BSL3predicted amino acid sequence (amino acids 1-273; SEQ ID NO:15); aminoacids 1-219 contain the predicted ECD.

FIGS. 6A-6B illustrate the nucleotide and predicted amino acid sequenceof the of the BSL3-Ig fusion construct. FIG. 6A shows the nucleotidesequence of BSL3-Ig (L232-6; SEQ ID NO:16): nucleotides 1-3 contain thetranslation initiation signal (ATG); nucleotides 1-651 encode the nativeBSL3 ECD; nucleotides 652-654 encode an artificial sequence introducedduring construction; nucleotides 655-1356 encode the Fc domain of humanIgG. FIG. 6B shows the predicted amino acid sequence of BSL3-Ig (L232-6;SEQ ID NO:17); amino acids 1-217 contain the BSL3 ECD; amino acid 218represents an artificial residue introduced during construction; aminoacids 219-451 contain the Fc domain of human IgG.

FIGS. 7A-7H illustrate the reagents and results of expression analysisperformed for BSL1, BSL2, and BSL3. FIG. 7A shows the nucleotidesequence of the BSL1 probe (SEQ ID NO:18) used for Northern blotanalysis. FIG. 7B shows the nucleotide sequence of the BSL2 probe (SEQID NO:19). FIG. 7C shows the nucleotide sequence of the BSL3 probe (SEQID NO:20). FIG. 7D shows the levels of BSL1, BSL3, and BSL3 mRNAobserved in various cell types as determined by Northern blot analysis;“PBT” indicates peripheral blood T-cells; “CD3/CD28” indicatesstimulation with anti-CD3 and anti-CD28 antibodies; “PMA” indicatesstimulation with phorbol 12 myristate 13 acetate; “LPS” indicatesstimulation with lipopolysaccharide; “PBM” indicates peripheral bloodmonocytes; “PHA” indicates stimulation with phytohemaglutinin;“GM-CSF/IL-4” indicates stimulation with GM-CSF and IL-4; “HMVEC”indicates human microvascular endothelial cells; “TNF-alpha” indicatesstimulation with TNF-alpha; and “H292 (Starved) indicates serum starvedH292 cells. FIG. 7E shows BSL3 expression levels in various tissue typesas determined by Northern analysis of commercially available blots usingradiolabeled BSL3/KpnI+XbaI probe. FIG. 7F shows BSL3 expression levelsin various tissue types as determined by hybridization analysis ofcommercially available microarrays using radiolabeled BSL3/KpnI+XbaIprobe. FIG. 7G shows BSL1 expression levels in various tissue types asdetermined by quantitative PCR. FIG. 7H shows BSL3 expression levels invarious tissue types as determined by quantitative PCR.

FIGS. 8A-8E illustrate the results of PCR analysis performed todetermine the relative levels of the BSL2-4616811 (BSL2vcvc) orBSL2-L165-35b (BSL2v1c2) transcripts in various cell types, with orwithout stimulation. The top arrow points to the bands representing theBSL2-4616811 transcript; the bottom arrow points to the bandsrepresenting the BSL2-L165-35b transcript. FIG. 8A shows the results forRAJI, RAMOS, PM-LCL, and PL-LCL cell types, with or without PMA andionomycin stimulation. FIG. 8B shows the results for CE-LCL cells, HL60,Thp1, and HUVEC cell types, with or without stimulation. FIG. 8C showsthe results for peripheral blood T-cells with or without PMA andionomycin stimulation. The results from cells isolated from two separatedonors are shown (donor 079 and donor 124). FIG. 8D shows the resultsfor CEM and HUT78 cells, with or without PMA and ionomycin stimulation.FIG. 8E shows the results of a PCR reaction using BSL2-4616811 plasmidas template. Lane 1: Lambda BstEII DNA ladder; lane 2: PCR product. Theresults demonstrate that the forward primer preferentially binds thespecific binding site in the first variable fold rather than for thehomologous site in the second variable fold of BSL2-4616811.

FIGS. 9A-9F illustrate the results of fluorescence activated cellsorting (FACS) performed using anti-BSL1, anti-BSL2, and anti-BSL3monoclonal antibodies (MAbs). FIG. 9A shows FACS analysis of A549epithelial lung cells using anti-BSL1 MAb. Column 1: no MAb; column 2:isotype control; column 3: BSL1 hybridoma supernatant 32. FIG. 9B showsFACS analysis of A549 epithelial lung cells using anti-BSL2-4616811 MAb.Column 1: no MAb; column 2: isotype control; column 3: anti-BSL2 MAb1F7G2; column 4: anti-BSL2 MAb 2B10D7; column 5: anti-BSL2 MAb 3E6D3;column 6: anti-BSL2 MAb 4C2C6; column 7: anti-BSL2 MAb 5D7E2. FIG. 9Cshows FACS analysis of various cell types using anti-BSL3 MAb. FIG. 9Dshows FACS analysis of human umbilical vein endothelial cells (HUVEC)with or without TNF-alpha stimulation using anti-BSL3 MAb. FIGS. 9E-9Fshow FACS analysis of peripheral blood monocytes (PBMC) with or withoutGM-CSF/IL-4 or PHA stimulation using anti-BSL3 MAb. FIG. 9E showsresults from cells isolated from donor 126; FIG. 9F shows results fromcells-isolated from donor-145.

FIGS. 10A-10D illustrate co-stimulation of peripheral blood T-cellsusing BSL3-Ig fusion proteins in the presence of anti-CD3 monoclonalantibody. The L6-Ig fusion protein is used as a negative control. FIG.10A shows results from cells isolated from donor 78. FIG. 10B showresults from cells isolated from donor 124. FIGS. 10C-10D show theresults from cells stimulated with anti-CD3 MAb and BSL3-Ig fusionprotein and blockaded with anti-BSL3 MAb. Column 1: anti-BSL3-1A4A1 MAb;column 2: anti-BSL3-2B6H7 MAb; and column 3: isotype control antibody.FIG. 10C shows results from cells isolated from donor 010; FIG. 10Dshows results from cells isolated from donor 127.

FIGS. 11A-11J illustrate suppression of peripheral blood T-cellproliferation using BSL2-4616811-Ig (BSL2vcvc-Ig) and/or anti-BSL2 MAb.FIG. 11A shows results obtained using decreasing concentrations ofanti-CD3 MAb, and constant concentrations of BSL2-4616811-Ig(BSL2vcvc-Ig) or ChiL6 fusion proteins. FIG. 11B shows results obtainedusing a constant concentration of anti-CD3 MAb, and decreasingconcentrations of BSL2-4616811-Ig (BSL2vcvc-Ig) or ChiL6 fusionproteins. FIG. 11C shows results obtained using a decreasingconcentration of anti-CD28 MAb, a constant concentration of anti-CD3MAb, and a constant concentration of BSL2-4616811-Ig (BSL2vcvc-Ig) orChiL6 fusion proteins. FIG. 11D shows results obtained using a constantconcentration of anti-CD3 MAb, a decreasing concentration ofBSL2-4616811-Ig (BSL2vcvc-Ig), BSL2-L165-35b-Ig (BSL2v1c2-Ig), or ChiL6fusion proteins. In the graph, “BSL2vcIg” represents BSL2-L165-35b-Ig(BSL2v1c2-Ig). FIG. 11E shows results obtained using a constantconcentration of anti-CD3 MAb, a constant concentration ofBSL2-4616811-Ig (BSL2vcvc-Ig) fusion protein, and decreasingconcentrations of anti-BSL2-1 MAb, anti-BSL2-5 MAb, or non-specific3_(—)15 MAb. FIG. 11F shows results obtained using a constantconcentration of anti-CD3 MAb, a constant concentration ofBSL2-4616811-Ig (BSL2vcvc-Ig) fusion protein, and decreasingconcentrations of anti-BSL2-1 MAb; anti-BSL-2-2 MAb, anti-BSL2-3 MAb,anti-BSL2-4 MAb, BSL2-5 MAb, or non-specific 3_(—)15 MAb. FIG. 11G showsresults obtained using a constant concentration of anti-CD3 MAb, aconstant concentration of ChiL6 fusion protein, and decreasingconcentrations of anti-BSL2-1 MAb or non-specific 3_(—)15 MAb, or noMAb. FIG. 11H shows results obtained using a constant concentration ofanti-CD3 MAb, a constant concentration of BSL2-L165-35b-Ig (BSL2v1c2-Ig)fusion protein, and decreasing concentrations of anti-BSL2-1 MAb ornon-specific 3_(—)15 MAb, or no MAb. FIG. 111 shows results obtainedusing a constant concentration of anti-CD3 MAb, followed by a constantconcentration of BSL2-4616811-Ig (BSL2vcvc-Ig) fusion protein, laterfollowed by decreasing concentrations of plate-bound anti-BSL2-1 MAb ornon-specific 3_(—)15 MAb, or no MAb. In this experiment, MAb was boundto the plate after addition of BSL2-4616811-Ig (BSL2vcvc-Ig). FIG. 11Jshows results obtained using a constant concentration of anti-CD3 MAb,followed by decreasing concentrations of plate-bound anti-BSL2-1 MAb,non-specific 3_(—)15 MAb, or no MAb, later followed by a constantconcentration of BSL2-4616811-Ig (BSL2vcvc-Ig) fusion protein. In thisexperiment, MAb was bound to the plate before addition ofBSL2-4616811-Ig (BSL2vcvc-Ig).

FIGS. 12A-12B illustrate results obtained from mixed lymphocytereactions. FIG. 12A shows reactions from cells incubated with decreasingconcentrations of BSL2-4616811-Ig (BSL2vcvc-Ig), CTLA-4-Ig, or ChiL6fusion proteins. In the graph, “(124×051)” indicates that T-cells fromdonor 124 were used as responders and monocytes from donor 051 were usedas stimulators for the reactions. FIG. 12B shows reactions from cellsincubated with BSL2-4616811-Ig (BSL2vcvc-Ig), BSL2-L165-35b-Ig(BSL2v1c2-Ig), or ChiL6 fusion proteins. In the graph, “BSL2vcIg”represents BSL2-L165-35b-Ig (BSL2v1c2-Ig) fusion protein; and “(82×148)”indicates that T-cells from donor 82 were used as responders andmonocytes from donor 148 were used as stimulators for the reactions.

FIG. 13 shows the results of a binding comparison of anti-BSL2 MAb toBSL2-4616811-Ig (BSL2vcvc-Ig) and BSL2v1c2-Ig. In the graph, “vcvc”represents BSL2-4616811-Ig (BSL2vcvc-Ig) fusion protein; “vc” representsBSL2-L165-35b-Ig (BSL2v1c2-Ig) fusion protein.

DETAILED DESCRIPTION OF THE INVENTION

Identification of B7-Related Factors

In accordance with the methods of the present invention, threeB7-related factors, designated BSL1, BSL2, and BSL3, have beenidentified and characterized. In addition, three distinct BSL2 splicevariants have been identified, including BSL2-4616811 (BSL2vcvc),BSL2-L165-21 (BSL2v2c2), and BSL2-L165-35b (BSLv1c2). These B7-relatedfactors may provide a molecular basis for the activation of immune orinflammatory response cells, such as T-cells, at different times and indifferent illnesses and disease states. In addition, the disclosedB7-related factors can be utilized in the prevention or treatmentcertain diseases by modulating the activity of immune or inflammatoryresponse cells, such as T-cells, using the methods described in detailherein. These methods can be used as prophylaxis or treatments forcancers or immune-related disorders as detailed below.

Notably, experiments described herein demonstrate that BSL2-4616811-Ig(BSL2vcvc-Ig) fusion protein acts synergistically with anti-BSL2 MAbs toinhibit T-cell proliferation. Accordingly, compositions comprisingBSL2-4616811-Ig (BSL2vcvc-Ig) fusion protein may be used alone or inconjunction with compositions comprising anti-BSL2 MAbs for treatmentsof various disorders, including acute and chronic transplant rejection,rheumatoid arthritis, multiple sclerosis, psoriasis, or other diseasesdescribed in detail herein. In addition, such compositions can be usedindividually or in combination for therapeutic applications such asxenotransplantation.

Identification of B7-related genes from cDNA libraries: To identifyB7-related factors, cDNA libraries can be constructed and analyzed usingseveral well-established techniques. Messenger RNA can be obtained fromcells expressing B7-1 and/or B7-related factors. For example, mRNA canbe obtained from differentiated human peripheral blood mononuclearcells. Alternatively, mRNA can be obtained from various subsets ofneoplastic B cells, including tumor cells isolated from patients withnon-Hodgkin's lymphoma (L. Chaperot et al. (1999) Exp. Hematol.27:479-88). Such cells are known to express B7-1 and, thus, may expressB7-related factors, and can also serve as a source of the mRNA forconstruction of the cDNA library.

Total cellular mRNA can be isolated by a variety of techniques, e.g.,guanidinium-thiocyanate extraction (J. M. Chirgwin et al. (1979)Biochemistry 18:5294-5299; Chomczynski et al. (1987) Anal. Biochem.162:156-9). Following isolation, poly(A)⁺ RNA can be purified usingoligo(dT) cellulose. The purified poly(A)⁺ RNA can then be used as atemplate for cDNA synthesis utilizing reverse transcriptase polymerasechain reaction (RT-PCR; see C. R. Newton et al. (1997) PCR 2^(nd) Ed,Scientific Publishers, Oxford, England). Following reversetranscription, the cDNA can be converted to double stranded DNA usingconventional techniques (see H. Okayama et al. (1982) Mol. Cell. Biol.2:161; U. Gubler et al. (1983) Gene 25:263).

Cloning of the double stranded cDNAs can be accomplished usingtechniques that are well known in the art (see J. Sambrook et al. (1989)Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y.; F. M. Ausubel et al. (1989) Current Protocols inMolecular Biology, John Wiley & Sons, New York, N.Y.). The use ofsynthetic adaptors prior to cloning is particularly preferred, since itobviates the need for cleavage of the cDNA with one or more restrictionenzymes (see, for example, E. C. Bottger (1989) Biotechniques 7:925-6,928-90). Using this method, non-self complementary, kinased adaptors canbe added to the DNA prior to ligation with the vector. Virtually anyadaptor can be employed.

A cDNA library sequence can be expressed when placed in the senseorientation in a vector that supplies an appropriate promoter. Vectorsmay also include an origin of replication and various enhancersequences, splice acceptor/donor sequences, and polyadenylationsequences. Vectors may further include a marker that allows forselection of cells containing the vector construct. Markers may be aninducible or non-inducible gene and will generally allow for positiveselection under induction, or without induction, respectively. Examplesof marker genes include neomycin, dihydrofolate reductase, glutaminesynthetase, and the like. Notably, prepared cDNA libraries can beobtained from various commercial sources (e.g., Incyte Genomics, Inc.,St. Louis, Mo.; Stratagene, La Jolla, Calif.)

The cDNA library can be used to clone B7-related factors utilizingexpression cloning techniques (see B. Seed et al. (1987) Proc. Natl.Acad. Sci. USA 84:3365-3369; A. Aruffo et al. (1987) Proc. Natl. Acad.Sci. USA 84:8573-8577). In one embodiment, plasmid DNA is introducedinto a cell line by known methods of transfection (e.g., DEAE-Dextran)and allowed to replicate and express the cDNA inserts. B7-1 antigen isdepleted from the transfected cells using an anti-B7-1 monoclonalantibody (e.g., 133 and B1.1) and anti-murine IgG and IgM coatedimmunomagnetic beads. Transfectants expressing B7-related factors arepositively selected by incubation with CTLA-4-Ig and CD28-Ig followed bypanning with anti-human Ig immunoglobulin. After panning, episomal DNAis recovered from the panned cells and transfected into a competentbacterial host, preferably Escherichia coli (E. coli). Plasmid DNA issubsequently reintroduced into the cell line and the cycle of expressionand panning repeated at least two times. Following the final panningcycle, plasmid DNA is prepared from individual colonies, transfectedinto the cell line and analyzed for expression of the B7-relatedpolypeptides by indirect immunofluorescence with CTLA-4-Ig and CD28-Ig.After cloning, plasmids are prepared from the clones strongly reactivewith the CTLA-4-Ig, and then sequenced using conventional sequencingtechniques (reviewed in G. W. Slater et al. (1998) Electrophoresis19:1525-41).

Identification of B7-related genes in protein sequence databases:Alternatively, B7-related factors can be identified by screeningavailable sequence databases. The polypeptide sequence encoded by apreviously identified B7 factor (e.g., B7-1, B7-2, or B7-H1) or aB7-related factor disclosed herein (e.g., BSL1, BSL2, or BSL3), can becompared with the polypeptide sequences present in various proteindatabases. Publicly available protein sequence databases, e.g., GenBank,GenPept, SWISS-PROT, Protein Data Bank (PDB), Protein InformationResource (PIR), Human UniGene (National Center for BiotechnologyInformation), can be used to determine if additional B7-related factorsare present in mammalian, preferably human, species. Alternatively,privately owned protein sequence databases, e.g., the Incyte Genomicssequence database (Incyte Genomics), can be used to identify B7-relatedfactors. Databases with relatively few redundant sequences, e.g., PIR orSWISS-PROT databases, can be used to improve the statisticalsignificance of a sequence match. However, databases which are morecomprehensive and up-to-date, e.g., GenBank, GenPept, and IncyteGenomics sequence databases (Incyte Genomics), are preferred.

Any method known in the art can be used to align and compare thepreviously identified B7 factor sequence with the sequences present inthe protein sequence databases. Preferably, the BLAST program is used(S. F. Altschul et al. (1990) J. Mol. Biol. 215:403-410; S. Karlin etal. (1990) Proc. Natl. Acad. Sci. USA 87:2264-68; S. Karlin et al.(1993) Proc. Natl. Acad. Sci. USA 90:5873-7). BLAST identifies localalignments between the sequence of the previously identified protein andthe protein sequences in the database, and predicts the probability ofthe local alignment occurring by chance. Although the original BLASTprograms utilized ungapped local alignments, more recently developedBLAST programs such as WU-BLAST2/BLAST v2.0 (S. F. Altschul et al.(1996) Methods Enzymol. 266, 460-480) have been modified to incorporategapped local alignments similar to SSEARCH (T. F. Smith et al. (1981) J.Mol. Biol. 147:195-197) and FASTA programs (W. R. Pearson (1990) MethodsEnzymol. 183:63-98). In addition, position-specific-iterated BLAST(PSI-BLAST) programs have been developed to identify weak butbiologically relevant sequence similarities (S. F. Altschul et al.(1997) Nucleic Acids Res. 25:3389-3402). Furthermore,pattern-hit-initiated BLAST (PHI-BLAST) programs have been designed toidentify specific patterns or sequence motifs shared bydistantly-related proteins (Z. Zhang et al. (1998) Nucleic Acids Res.26:3986-3990). Specialized BLAST programs are also available forperforming searches of human, microbial, and malaria genome sequences,as well as searches for vector, immunoglobulin, and predicted humanconsensus sequences (National Center for Biotechnology Information(NCBI), Bethesda, Md.).

Both FASTA and BLAST programs identify very short exact sequence matchesbetween the query sequence and the databases sequences, analyze the bestshort sequence matches (“hits”) to determine if longer stretches ofsequence similarity are present, and then optimize the best hits bydynamic programming (S. F. Altschul et al. (1990) J. Mol. Biol.215:403-410; W. R. Pearson, supra). In contrast, the SSEARCH programcompares the query sequence to all the sequences in the database viapair-wise sequence comparisons (T. F. Smith et al., supra). Thus, theSSEARCH program is considered more sensitive than the BLAST and FASTAprograms, but it is also significantly slower. The BLAST and FASTAprograms utilize several approximations to increase their searchingspeed, and utilize statistical parameters (see below) to increasesensitivity and selectivity to approximate the performance of theSSEARCH program. A particular sequence alignment program can be chosenbased on the requirements of a sequence search, or individualpreferences. In some cases, it may be necessary to use more than onesearch alignment program to confirm search alignment results or resolveambiguous search results.

Typically, BLAST analysis employs (i) a scoring matrix (such as, e.g.,BLOSSUM 62 or PAM 120) to assign a weighted homology value to eachresidue and (ii) a filtering program(s) (such as SEG or XNU) thatrecognizes and eliminates highly repeated sequences from thecalculation. An appropriate homology cutoff is then determined byperforming BLAST comparisons (using a particular scoring matrix andfiltering program) between sequences that are known to be related. Itwill be understood that other appropriate scoring matrices and filteringprograms may be used when the cutoff is calibrated as described herein.That is, the particular cutoff point may vary when different standardparameters are used, but it will correspond to the P(N) scores exhibitedwhen highly related sequences are compared using those particularparameters.

B7-Related Nucleic Acids

One aspect of the present invention pertains to isolated nucleic acidshaving a nucleotide sequence such as BSL1 (e.g., SEQ ID NO:1 or 3), BSL2(e.g., SEQ ID NO:6, 10, 12, or 131), or BSL3 (e.g., SEQ ID NO:14), orfragments thereof. The nucleic acid molecules of the invention can beDNA or RNA. A preferred nucleic acid is a DNA encoding the human BSL1(e.g., SEQ ID NO:2), BSL2 (e.g., SEQ ID NO:7, 11, or 13), or BSL3 (e.g.,SEQ ID NO:15), or fragments or functional equivalents thereof. Suchnucleic acids can comprise at least 15, 20, 25, 50, 60, 100, 200, 240,255, 270, 300, 305, 310, 410, 500, 619, 630, 700, or 1000 contiguousnucleotides.

The term “isolated” as used throughout this application refers to aB7-related nucleic acid, polypeptide, peptide, protein fusion, orantibody, that is substantially free of cellular material or culturemedium. An isolated or substantially purified molecule contains lessthan about 50%, preferably less than about 25%, and most preferably lessthan about 10%, of the cellular components with which it was associated.

The term “functional equivalent” is intended to include nucleotidesequences encoding functionally equivalent B7-related factors. Afunctional equivalent of a B7-related protein includes fragments orvariants that perform at least one characteristic function of theB7-related protein (e.g., ligand-binding, antigenic, intra-, orintercellular activity). For example, DNA sequence polymorphisms withinthe nucleotide sequence of a B7-related factor, especially those withinthe third base of a codon, may result in “silent” mutations, which donot affect the encoded amino acid sequence of the protein due to thedegeneracy of the genetic code.

In one embodiment, the present invention encompasses a polynucleotidecomprising the start codon and the remaining coding sequence ofBSL2-4616811 (BSL2vcvc). Specifically, the invention encompasses apolynucleotide comprising nucleotides 1 through 1602 of SEQ ID NO:131.The invention also encompasses a polynucleotide comprising nucleotides121 through 1722 of SEQ ID NO:6, and the corresponding polypeptidecomprising amino acids 1 through 534 of SEQ ID NO:7. Also encompassedare vectors comprising these polynucleotides, and host cells comprisingthese vectors.

In another embodiment, the present invention embraces a polynucleotidelacking the initiating start codon, but including the remaining codingsequence of BSL2-4616811 (BSL2vcvc). Specifically, the inventionembraces a polynucleotide comprising nucleotides 4 through 1602 of SEQID NO:131. In addition, the invention embraces a polynucleotidecomprising nucleotides 124 through 1722 of SEQ ID NO:6, and thepolypeptide corresponding to amino acids 2 through 534 of SEQ ID NO:7.Also embraced are vectors comprising these polynucleotides, and hostcells comprising these vectors.

The present invention also encompasses a polynucleotide comprising thestart codon and the remaining coding sequence of BSL2-L165-35b(BSL2v1c2). Specifically, the invention encompasses a polynucleotidecomprising nucleotides 1 through 948 of SEQ ID NO:12. The inventionfurther encompasses a corresponding polypeptide comprising amino acids 1through 316 of SEQ ID NO:13. Also encompassed are vectors comprisingthese polynucleotides, and host cells comprising these vectors.

The present invention also embraces a polynucleotide lacking theinitiating start codon, but including-the-remaining coding sequence ofBSL2-L165-35b (BSL2v1c2). Specifically, the invention embraces apolynucleotide comprising nucleotides 4 through 948 of SEQ ID NO:12. Inaddition, the invention embraces a polypeptide corresponding to aminoacids 2 through 316 of SEQ ID NO:13. Also embraced are vectorscomprising these polynucleotides, and host cells comprising thesevectors.

The invention further encompasses a polynucleotide comprising the startcodon and the remaining coding sequence of BSL2-L165-21 (BSL2v2c2).Specifically, the invention encompasses a polynucleotide comprisingnucleotides 1 through 948 of SEQ ID NO:10. The invention furtherencompasses a corresponding polypeptide comprising amino acids 1 through316 of SEQ ID NO:11. Also encompassed are vectors comprising thesepolynucleotides, and host cells comprising these vectors.

The present invention further embraces a polynucleotide lacking theinitiating start codon, but including the remaining coding sequence ofBSL2-L165-21 (BSL2v2c2). Specifically, the invention embraces apolynucleotide comprising nucleotides 4 through 948 of SEQ ID NO:10. Inaddition, the invention embraces a polypeptide corresponding to aminoacids 2 through 316 of SEQ ID NO:11. Also embraced are vectorscomprising these polynucleotides, and host cells comprising thesevectors.

Preferred embodiments include an isolated nucleic acid sharing at least60, 70, 80, 85, 90, 95, 97, 98, 99, 99.5, or 100% sequence identity witha polynucleotide sequence of BSL1 (e.g., SEQ ID NO:1 or 3), BSL2 (e.g.,SEQ ID NO:6, 10, 12, or 131), or BSL3 (e.g., SEQ ID NO:15). Thispolynucleotide sequence may be identical to the nucleotide sequence ofBSL1 (e.g., SEQ ID NO:1 and 3), BSL2 (e.g., SEQ ID NO:6, 10, 12, or131), or BSL3 (e.g., SEQ ID NO:14), or may include up to a certaininteger number of nucleotide alterations as compared to the referencesequence.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing. Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D. (1988) SIAM J. Applied Math.,48:1073.

For nucleic acids, sequence identity can be determined by comparing aquery sequences to sequences in publicly available sequence databases(NCBI) using the BLASTN2 algorithm (S. F. Altschul et al. (1997) Nucl.Acids Res., 25:3389-3402). The parameters for a typical search are:E=0.05, v=50, B=50, wherein E is the expected probability score cutoff,V is the number of database entries returned in the reporting of theresults, and B is the number of sequence alignments returned in thereporting of the results (S. F. Altschul et al. (1990) J. Mol. Biol.,215:403-410).

In another approach, nucleotide sequence identity can be calculatedusing the following equation: % identity=(number of identicalnucleotides)/(alignment length in nucleotides)*100. For thiscalculation, alignment length includes internal gaps but not terminalgaps. Alternatively, nucleotide sequence identity can be determinedexperimentally using the specific hybridization conditions describedbelow.

In accordance with the present invention, nucleic acid alterations areselected from the group consisting of at least one nucleotide deletion,substitution, including transition and transversion, insertion, ormodification (e.g., via RNA or DNA analogs, dephosphorylation,methylation, or labeling). Alterations may occur at the 5′ or 3′terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongthe nucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence. Alterations of a nucleic acidsequence of BSL1 (e.g., SEQ ID NO:1 and 3), BSL2 (e.g., SEQ ID NO:6, 10,12, or 131), or BSL3 (e.g., SEQ ID NO:14) may create nonsense, missense,or frameshift mutations in the coding sequence, and thereby alter thepolypeptide encoded by the nucleic acid.

Also encompassed by the present invention are splice variants derivedfrom the BSL1 (e.g., SEQ ID NO:1 and 3), BSL2 (e.g., SEQ ID NO:6, 10,12, or 131), or BSL3 (e.g., SEQ ID NO:14) nucleic acid sequences. Asused herein, the term “splice variant” refers to variant B7-relatednucleic acids and polypeptides produced by differential processing ofthe primary transcript(s) of genomic DNA. An alternate splice variantmay comprise, for example, any one of the sequences of BSL2 (e.g., SEQID NO:6, 10, 12, or 131) disclosed herein. Alternate splice variants canalso comprise other combinations of introns/exons of BSL1, BSL2, orBSL3, which can be determined by those of skill in the art. Alternatesplice variants can be determined experimentally, for example, byisolating and analyzing cellular RNAs (e.g., Southern blotting or PCR),or by screening cDNA libraries using the B7-related nucleic acid probesor primers described herein. In another approach, alternate splicevariants can be predicted using various methods, computer programs, orcomputer systems available to practitioners in the field.

General methods for splice site prediction can be found in Nakata (1985)Nucleic Acids Res. 13:5327-5340. In addition, splice sites can bepredicted using, for example, the GRAIL™ (E. C. Uberbacher and R. J.Mural (1991) Proc. Natl. Acad. Sci. USA, 88:11261-11265; E. C.Uberbacher (1995) Trends Biotech., 13:497-500; GenView (L. Milanesi etal. (1993) Proceedings of the Second International Conference onBioinformatics, Supercomputing, and Complex Genome Analysis, H. A. Limet al. (eds), World Scientific Publishing, Singapore, pp. 573-588;SpliceView; and HSPL (V. V. Solovyev et al. (1994) Nucleic Acids Res.22:5156-5163; V. V. Solovyev et al. (1994) “The Prediction of HumanExons by Oligonucleotide Composition and Discriminant Analysis ofSpliceable Open Reading Frames,” R. Altman et al. (eds), The SecondInternational conference on Intelligent systems for Molecular Biology,AAAI Press, Menlo Park, Calif., pp. 354-362; V. V. Solovyev et al.(1993) “Identification Of Human Gene Functional Regions Based OnOligonucleotide Composition,” L. Hunter et al. (eds), In Proceedings ofFirst International conference on Intelligent System for MolecularBiology, Bethesda, pp. 371-379) computer systems.

Additionally, computer programs such as GeneParser (E. E. Snyder and G.D. Stormo (1995) J. Mol. Biol. 248: 1-18; E. E. Snyder and G. D. Stormo(1993) Nucl. Acids Res. 21(3): 607-613; MZEF (M. Q. Zhang (1997) Proc.Natl. Acad. Sci. USA, 94:565-568; MORGAN (S. Salzberg et al. (1998) J.Comp. Biol. 5:667-680; S. Salzberg et al., eds. (1998) ComputationalMethods in Molecular Biology, Elsevier Science, New York, N.Y., pp.187-203); VEIL (J. Henderson et al. (1997) J. Comp. Biol. 4:127-141);GeneScan (S. Tiwari et al. (1997) CABIOS (BioInformatics) 13: 263-270);GeneBuilder (L. Milanesi et al. (1999) Bioinformatics 15:612-621);Eukaryotic GeneMark (J. Besemer et al. (1999) Nucl. Acids Res.27:3911-3920); and FEXH (V. V. Solovyev et al. (1994) Nucl. Acids Res.22:5156-5163) can be used. In addition, splice sites (i.e., former orpotential splice sites) in cDNA sequences can be predicted using, forexample, the RNASPL (V. V. Solovyev et al. (1994) Nucl. Acids Res.22:5156-5163); or INTRON (A. Globek et al. (1991) INTRON version 1.1manual, Laboratory of Biochemical Genetics, NIMH, Washington, D.C.)programs.

The present invention also encompasses naturally-occurring polymorphismsof BSL1 (e.g., SEQ ID NO:1 and 3), BSL2 (e.g., SEQ ID NO:6, 10, 12, or131), or BSL3 (e.g., SEQ ID NO:14). As will be understood by those inthe art, the genomes of all organisms undergo spontaneous mutation inthe course of their continuing evolution generating variant forms ofgene sequences (Gusella (1986) Ann. Rev. Biochem. 55:831-854).Restriction fragment length polymorphisms (RFLPs) include variations inDNA sequences that alter the length of a restriction fragment in thesequence (Botstein et al. (1980) Am. J. Hum. Genet. 32, 314-331. RFLPshave been widely used in human and animal genetic analyses (see WO90/13668; WO 90/11369; Donis-Keller (1987) Cell 51:319-337; Lander etal. (1989) Genetics 121: 85-99). Short tandem repeats (STRs) includetandem di-, tri- and tetranucleotide repeated motifs, also termedvariable number tandem repeat (VNTR) polymorphisms. VNTRs have been usedin identity and paternity analysis (U.S. Pat. No. 5,075,217; Armour etal. (1992) FEBS Lett. 307:113-115; Horn et al., WO 91/14003; Jeffreys,EP 370,719), and in a large number of genetic mapping studies.

Single nucleotide polymorphisms (SNPs) are far more frequent than RFLPS,STRs, and VNTRs. SNPs may occur in protein coding (e.g., exon), ornon-coding (e.g., intron, 5′UTR, 3′UTR) sequences. SNPs in proteincoding regions may comprise silent mutations that do not alter the aminoacid sequence of a protein. Alternatively, SNPs in protein codingregions may produce conservative or non-conservative amino acid changes,described in detail below. In some cases, SNPs may give rise to theexpression of a defective or other variant protein and, potentially, agenetic disease. SNPs within protein-coding sequences can give rise togenetic diseases, for example, in the β-globin (sickle cell anemia) andCFTR (cystic fibrosis) genes. In non-coding sequences, SNPs may alsoresult in defective protein expression (e.g., as a result of defectivesplicing). Other single nucleotide polymorphisms have no phenotypiceffects.

Single nucleotide polymorphisms can be used in the same manner as RFLPsand VNTRs, but offer several advantages. Single nucleotide polymorphismstend to occur with greater frequency and are typically spaced moreuniformly throughout the genome than other polymorphisms. Also,different SNPs are often easier to distinguish than other types ofpolymorphisms (e.g., by use of assays employing allele-specifichybridization probes or primers). In one embodiment of the presentinvention, a BSL1 (e.g., SEQ ID NO:1 and 3), BSL2 (e.g., SEQ ID NO:6,10, 12, or 131), or BSL3 (e.g., SEQ ID NO:14) nucleic acid contains atleast one SNP. Various combinations of these SNPs are also encompassedby the invention. In a preferred aspect, a B7-related SNP is associatedwith a immune system disorder, such as the disorders described in detailherein.

Further encompassed by the present invention are nucleic acid moleculesthat share moderate homology with the BSL1 (e.g., SEQ ID NO:1 and 3),BSL2 (e.g., SEQ ID NO:6, 10, 12, or 131), or BSL3 (e.g., SEQ ID NO:14)nucleic acid sequences, and hybridize to the BSL1, BSL2, or BSL3 nucleicacid molecules under moderate stringency hybridization conditions. Morepreferred are nucleic acid molecules that share substantial homologywith the BSL1, BSL2, or BSL3 nucleic acid sequences and hybridize to theBSL1, BSL2, or BSL3 nucleic acid molecules under high stringencyhybridization conditions. As used herein, the phrase “moderate homology”refers to sequences which share at least 60% sequence identity with areference sequence (e.g., BSL1, BSL2 or BSL3), whereas the phrase“substantial homology” refers to sequences that share at least 90%sequence identity with a reference sequence. It is recognized, however,that polypeptides and the nucleic acids encoding such polypeptidescontaining less than the above-described level of homology arising assplice variants or that are modified by conservative amino acidsubstitutions (or substitution of degenerate codons) are contemplated tobe within the scope of the present invention.

The phrase “hybridization conditions” is used herein to refer toconditions under which a double-stranded nucleic acid hybrid is formedfrom two single nucleic acid strands, and remains stable. As known tothose of skill in the art, the stability of the hybrid sequence isreflected in the melting temperature (T_(m)) of the hybrid (see F. M.Ausubel et al., Eds, (1995) Current Protocols in Molecular Biology, JohnWiley and Sons, Inc., New York, N.Y.). The T_(m) decreases approximately0.5° C. to 1.5° C. with every 1% decrease in sequence homology. Ingeneral, the stability of a hybrid sequence is a function of the lengthand guanine/cytosine content of the hybrid, the sodium ionconcentration, and the incubation temperature. Typically, thehybridization reaction is initially performed under conditions of lowstringency, followed by washes of varying, but higher, stringency.Reference to hybridization stringency relates to such washingconditions.

In accordance with the present invention, “high stringency” conditionscan be provided, for example, by hybridization in 50% formamide, 5×Denhardt's solution, 5×SSPE, and 0.2% SDS at 42° C., followed by washingin 0.1×SSPE and 0.1% SDS at 65° C. By comparison, “moderate stringency”can be provided, for example, by hybridization in 50% formamide, 5×Denhardt's solution, 5×SSPE, and 0.2% SDS at 42° C., followed by washingin 0.2×SSPE and 0.2% SDS at 65° C. In addition, “low stringency”conditions can be provided, for example, by hybridization in 10%formamide, 5× Denhardt's solution, 6×SSPE, and 0.2% SDS at 42° C.,followed by washing in 1×SSPE and 0.2% SDS at 50° C. It is understoodthat these conditions may be varied using a variety of buffers andtemperatures well known to those skilled in the art.

In a preferred embodiment of the present invention, the nucleic acid isa DNA molecule encoding at least a portion of the B7-related factor. Anucleic acid molecule encoding a novel B7-related factor can be obtainedfrom mRNA present in activated B lymphocytes. It may also be possible toobtain nucleic acid molecules encoding B7-related factors from B cellgenomic DNA. Thus, a nucleic acid encoding a B7-related factor can becloned from either a cDNA or a genomic library in accordance with theprotocols described in detail herein. Nucleic acids encoding novelB7-related factors can also be cloned from genomic DNA or cDNA usingestablished polymerase chain reaction (PCR) techniques (see K. Mullis etal. (1986) Cold Spring Harbor Symp. Quant. Biol. 51:260; K. H. Roux(1995) PCR Methods Appl. 4:S185) in accordance with the nucleic acidsequence information provided herein. The nucleic acid molecules of theinvention, or fragments thereof, can also be chemically synthesizedusing standard techniques. Various methods of chemically synthesizingpolydeoxynucleotides are known, including solid-phase synthesis which,like peptide synthesis, has been fully automated in commerciallyavailable DNA synthesizers (see, for example, U.S. Pat. No. 4,598,049 toItakura et al.; U.S. Pat. No. 4,458,066 to Caruthers et al.; U.S. Pat.Nos. 4,401,796 and 4,373,071 to Itakura).

It will be appreciated by one skilled in the art that variations in oneor more nucleotides (up to about 3-4% of the nucleotides) of the nucleicacid molecules encoding novel B7-related factors may exist amongindividuals within a population due to natural allelic variation. Anyand all such nucleotide variations and resulting amino acidpolymorphisms are within the scope of the invention. Furthermore, theremay be one or more isoforms or related, cross-reacting family members ofthe B7-related factors described herein. Such isoforms or family membersare defined as polypeptides that are related in function and amino acidsequence to a B7-related factor (e.g., BSL1, BSL2, or BSL3), but encodedby genes at different loci. In addition, it is possible to modify theDNA sequence of B7-related factors using genetic techniques to produceproteins or peptides with altered amino acid sequences.

DNA sequence mutations can be introduced into a nucleic acid encoding aB7-related factor by any one of a number of methods, including those forproducing simple deletions or insertions, systematic deletions,insertions or substitutions of clusters of bases or substitutions ofsingle bases, to generate desired variants. Mutations of the B7-relatednucleic acid molecule to generate amino acid substitutions or deletionsare preferably obtained by site-directed mutagenesis. Site directedmutagenesis systems are well known in the art, and can be obtained fromcommercial sources (see, for example, Amersham Pharmacia Biotech, Inc.,Piscataway, N.J.). Guidance in determining which amino acid residues maybe substituted, inserted, or deleted without abolishing biological orimmunological activity may be found using computer programs well knownin the art, for example, DNASTAR software (DNASTAR, Inc., Madison,Wis.). Mutant forms of the BSL1 (e.g., SEQ ID NO:1 and 3), BSL2 (e.g.,SEQ ID NO:6, 10, 12, or 131), or BSL3 (e.g., SEQ ID NO:14) nucleic acidmolecules are considered within the scope of the present invention,where the expressed polypeptide or peptide is capable modulating theactivity and/or proliferation of immune or inflammatory cells (e.g.,T-cells).

A fragment of the nucleic acid molecule encoding a novel B7-relatedfactor is defined as a nucleotide sequence having fewer nucleotides thanthe nucleotide sequence encoding the entire amino acid sequence of theB7-related factor. Nucleic acid fragments which encode polypeptideswhich retain the ability to bind to their natural ligand(s) on immune orinflammatory response cells, such as T-cells, and either amplify orblock immune responses (as evidenced by, for example, lymphokineproduction and/or T-cell proliferation by T-cells that have received aprimary activation signal) are considered within the scope of theinvention. For example, nucleic acid fragments that encode polypeptidesor peptides of a B7-related factor that retain the ability of thepolypeptides or peptides to bind CD28/CTLA-4 and/or CD28-/CTLA-4-relatedligand(s) and deliver a modulatory (e.g., co-stimulatory or inhibitory)signal to T-cells are within the scope of the invention. Generally, thenucleic acid molecule encoding a fragment of a B7-related factor will beselected from the coding sequence for the mature protein. However, insome instances it may be desirable to select all or part of a fragmentor fragments from the coding region that includes the leader sequence.

In one embodiment of the present invention, a nucleic acid moleculecorresponding to a fragment of a BSL1 (e.g., SEQ ID NO:1 or 3), BSL2(e.g., SEQ ID NO:6, 10, 12, or 131), or BSL3 (e.g., SEQ ID NO:14)nucleic acid sequence can be used as a probe for assaying a biologicalsample or the expression of one or more B7-related factors, or as aprimer for DNA sequencing or PCR amplification. Preferably, suchfragments are at least 8 contiguous nucleotides in length, morepreferably at least 12 contiguous nucleotides in length, even morepreferably at least 15 contiguous nucleotides in length, and even morepreferably at least 20 contiguous nucleotides in length. Nucleic acidmolecules within the scope of the invention may also contain linkersequences, modified restriction endonuclease sites, and other sequencesuseful for molecular cloning, expression, or purification of recombinantprotein or fragments thereof. Nucleic acid molecules in accordance withthe present invention may also be conjugated with radioisotopes, orchemiluminescent, fluorescent, or other labeling compounds (e.g.,digoxigenin). In addition, the nucleic acid molecules of the presentinvention may be modified by nucleic acid modifying enzymes, forexample, kinases or phosphatases. These and other modifications ofnucleic acid molecules are well known in the art.

In addition, a nucleic acid molecule that encodes a B7-related factor,or a biologically active fragment thereof, can be ligated to aheterologous sequence to encode a fusion protein (also called a chimericprotein). For example, it may be useful to construct a nucleic acidencoding a fusion protein comprising a B7-related factor and the Fcdomain of human IgG1 as described herein. In a preferred embodiment, theimmunoglobulin sequences used in construction of the BSL1, BSL2, or BSL3immunofusion proteins of the present innovation are obtained from anIgG1 immunoglobulin heavy chain domain. The resulting BSL1-Ig (e.g., SEQID NO:4), BSL2-Ig, (e.g., SEQ ID NO:8, SEQ ID NO:132, or SEQ NO:134),and BSL3-Ig (e.g., SEQ ID NO:17) fusion constructs can then be expressedin host cells, and used to prepare pharmaceutical compositions usefulfor immunomodulation (see below). Fusion proteins comprising B7-relatedpolypeptides can also be used for the isolation and purification ofB7-related polypeptides or antibodies (see below). In addition, fusionproteins can be used to identify cellular ligands or binding partnersfor BSL1, BSL2, or BSL3 (see below).

In one aspect, polynucleotides encoding 1) a natural or heterologoussignal sequence (ss); 2) a B7-related polypeptide (e.g., BSL1, BSL2, orBSL3) that lacks a signal sequence; and 3) an Fc domain can be clonedserially to produce a fusion protein that can be depicted as ss-BSL1-Ig,ss-BSL2-Ig, ss-BSL3-Ig. Alternately, polynucleotides encoding 1) anatural or heterologous signal sequence (ss); 2) an Fc domain; and 3) aB7-related polypeptide (e.g., BSL1, BSL2, or BSL3) that lacks a signalsequence can be cloned serially to produce a fusion protein that can bedepicted as ss-Ig-BSL1, ss-Ig-BSL2, ss-Ig-BSL3. Thus, B7-relatedpolypeptide for this invention may be fused to the C-terminus orN-terminus of the Fc domain. In addition, the polynucleotide sequencemay encode a proteolytic cleavage site positioned between the B7-relatedpolypeptide and the Fc domain, which can be used to separate theB7-related polypeptide from the Fc domain. Notably, a major advantage ofusing IgG1 for protein fusions is that IgG1 immunofusions can bepurified efficiently on immoblized protein A. However, other structuraland functional properties may be considered when choosing the Ig fusionpartner for a particular immunofusion construction.

B7-Related Nucleic Acid Expression Vectors

Another aspect of the present invention pertains to expression vectorscomprising a nucleic acid encoding at least one B7-related factor, asdescribed herein, operably linked to at least one regulatory sequence.“Operably linked” is intended to mean that the nucleotide acid sequenceis linked to a regulatory sequence in a manner that allows expression ofthe nucleotide sequence. Regulatory sequences are known in the art andare selected to direct expression of the desired protein in anappropriate host cell. Accordingly, the term regulatory sequenceincludes promoters, enhancers and other expression control elements (seeD. V. Goeddel (1990) Methods Enzymol. 185:3-7). It should be understoodthat the design of the expression vector may depend on such factors asthe choice of the host cell to be transfected and/or the type ofpolypeptide desired to be expressed.

Appropriate host cells for use with the present invention includebacteria, fungi, yeast, plant, insect, and animal cells, especiallymammalian and human cells. Preferred replication and inheritance systemsinclude M13, ColE1, SV40, baculovirus, lambda, adenovirus, CEN ARS, 2 μmARS and the like. Several regulatory elements (e.g., promoters) havebeen isolated and shown to be effective in the transcription andtranslation of heterologous proteins in the various hosts. Suchregulatory regions, methods of isolation, manner of manipulation, etc.are known in the art. Non-limiting examples of bacterial promotersinclude the β-lactamase (penicillinase) promoter; lactose promoter;tryptophan (trp) promoter; araBAD (arabinose) operon promoter;lambda-derived P₁ promoter and N gene ribosome binding site; and thehybrid tac promoter derived from sequences of the trp and lac UV5promoters. Non-limiting examples of yeast promoters include the3-phosphoglycerate kinase promoter, glyceraldehyde-3-phosphatedehydrogenase (GAPDH) promoter, galactokinase (GAL1) promoter,galactoepimerase promoter, and alcohol dehydrogenase (ADH1) promoter.Suitable promoters for mammalian cells include, without limitation,viral promoters, such as those from Simian Virus 40 (SV40), Rous sarcomavirus (RSV), adenovirus (ADV), and bovine papilloma virus (BPV).

Eukaryotic cells may also require terminator sequences, polyadenylationsequences, and enhancer sequences that modulate gene expression.Sequences that cause amplification of the gene may also be desirable.These sequences are well known in the art. Furthermore, sequences thatfacilitate secretion of the recombinant product from cells, including,but not limited to, bacteria, yeast, and animal cells, such as secretorysignal sequences and/or preprotein or proprotein sequences, may also beincluded. Such sequences are well described in the art.

Suitable expression vectors include, but are not limited to, pUC,pBluescript (Stratagene), pET (Novagen, Inc., Madison, Wis.), and pREP(Invitrogen) plasmids. Vectors can contain one or more replication andinheritance systems for cloning or expression, one or more markers forselection in the host, e.g. antibiotic resistance, and one or moreexpression cassettes. The inserted coding sequences can be synthesizedby standard methods, isolated from natural sources, or prepared ashybrids. Ligation of the coding sequences to transcriptional regulatoryelements (e.g., promoters, enhancers, and/or insulators) and/or to otheramino acid encoding sequences can be carried out using establishedmethods.

In one embodiment, the expression vector comprises a nucleic acidencoding at least a portion of the BSL1, BSL2, or BSL3 polypeptide. Inanother embodiment, the expression vector comprises a DNA sequenceencoding the B7-related factor and a DNA sequence encoding anotherB7-related factor or a heterologous polypeptide or peptide. Suchexpression vectors can be used to transfect host cells to therebyproduce polypeptides or peptides, including fusion proteins or peptidesencoded by nucleic acid molecules as described below.

Isolation of B7-Related Polypeptides

Yet another aspect of the present invention pertains to methods ofisolating B7-related polypeptides and related peptides. As used herein,the terms “protein” and “polypeptide” are synonymous. Peptides aredefined as fragments or portions of proteins or polypeptides, preferablyfragments or portions having the same or equivalent function or activityas the complete protein. Both naturally occurring and recombinant formsof the B7-related polypeptides or peptides may be used in assays andtreatments according to the present invention. Methods for directlyisolating and purifying polypeptides or peptides from natural sourcessuch as cellular or extracellular lysates are well known in the art (seeE. L. V. Harris and S. Angal, Eds. (1989) Protein Purification Methods:A Practical Approach, IRL Press, Oxford, England). Such methods include,without limitation, preparative disc-gel electrophoresis, isoelectricfocusing, high-performance liquid chromatography (HPLC), reversed-phaseHPLC, gel filtration, ion exchange and partition chromatography, andcountercurrent distribution, and combinations thereof. Naturallyoccurring polypeptides can be purified from many possible sources, forexample, plasma, body cells and tissues, or body fluids.

To produce recombinant B7-related polypeptides or peptides, DNAsequences encoding the B7-related polypeptides or peptides are clonedinto a suitable vector for expression in intact host cells or incell-free translation systems (see J. Sambrook et al., supra).Prokaryotic and eukaryotic vectors and host cells may be employed. Theparticular choice of a vector, host cell, or translation system is notcritical to the practice of the invention. DNA sequences can beoptimized, if desired, for more efficient expression in a given hostorganism. For example, codons can be altered to conform to the preferredcodon usage in a given host cell or cell-free translation system usingtechniques routinely practiced in the art.

For some purposes, it may be preferable to produce peptides orpolypeptides in a recombinant system wherein the peptides orpolypeptides carry additional sequence tags to facilitate purification.Such markers include epitope tags and protein tags. Non-limitingexamples of epitope tags include c-myc, haemagglutinin (HA),polyhistidine (6×-HIS; SEQ ID NO:93), GLU-GLU, and DYKDDDDK (FLAG®; SEQID NO:94) epitope tags. Epitope tags can be added to peptides by anumber of established methods. DNA sequences of epitope tags can beinserted as oligonucleotides or through primers used in PCRamplification into or adjacent to a coding sequence of interest. As analternative, a coding sequence of interest can be cloned into specificvectors that create fusions with epitope tags; for example, pRSETvectors (Invitrogen Corp., San Diego, Calif.). Non-limiting examples ofprotein tags include glutathione-S-transferase (GST), green fluorescentprotein (GFP), and maltose binding protein (MBP). Protein tags areattached to peptides or polypeptides by several well-known methods. Inone approach, the coding sequence of a polypeptide or peptide can becloned into a vector that creates a fusion between the polypeptide orpeptide and a protein tag of interest. Suitable vectors include, withoutlimitation, the exemplary plasmids, pGEX (Amersham Pharmacia Biotech,Inc., Piscataway, N.J.), pEGFP (CLONTECH Laboratories, Inc., Palo Alto,Calif.), and pMAL™ (New England BioLabs, Inc., Beverly, Mass.).Following expression, the epitope or protein tagged polypeptide orpeptide can be purified from a crude lysate of the translation system orhost cell by chromatography on an appropriate solid-phase matrix. Insome cases, it may be preferable to remove the epitope or protein tag(i.e., via protease cleavage) following purification.

Suitable cell-free expression systems for use in accordance with thepresent invention include rabbit reticulocyte lysate, wheat germextract, canine pancreatic microsomal membranes, E. coli S30 extract,and coupled transcription/translation systems (Promega Corp., Madison,Wis.). These systems allow the expression of recombinant polypeptides orpeptides upon the addition of cloning vectors, DNA fragments, or RNAsequences containing coding regions and appropriate promoter elements.

Host cells for recombinant cloning vectors include bacterial,archebacterial, fungal, plant, insect and animal cells, especiallymammalian cells. Of particular interest are E. coli, B. subtilis, S.aureus, S. cerevisiae, S. pombe, N. crassa, SF9, C129, 293, NIH 3T3,CHO, COS, and HeLa cells. Such cells can be transformed, transfected, ortransduced, as appropriate, by any suitable method includingelectroporation, CaCl₂—, LiCl—, LiAc/PEG-, spheroplasting-,Ca-Phosphate, DEAE-dextran, liposome-mediated DNA uptake, injection,microinjection, microprojectile bombardment, or other establishedmethods.

In order to identify host cells that contain the expression vector, agene that contains a selectable marker is generally introduced into thehost cells along with the gene of interest. Preferred selectable markersinclude those that confer resistance to drugs, such as G418, hygromycin,methotrexate, or ampicillin. Selectable markers can be introduced on thesame plasmid as the gene of interest. Host cells containing the gene ofinterest are identified by drug selection, as cells that carry thedrug-resistance marker survive in growth media containing thecorresponding drug.

The surviving cells can be screened for production of recombinantB7-related polypeptides, or peptides or fusions thereof. In oneembodiment, the recombinant polypeptides are secreted to the cellsurface, and can be identified by cell surface staining with ligands tothe B cell antigens (e.g., CD28-Ig). In another embodiment, therecombinant polypeptides are retained in the cytoplasm of the hostcells, and can be identified in cell extracts using anti-B7-relatedpolypeptide antibodies. In yet another embodiment, soluble recombinantpolypeptides are secreted into the growth media, and can be identifiedby screening the growth media with anti-B7-related polypeptideantibodies. A soluble, secreted recombinant B7-polypeptide includes theextracellular domain of the polypeptide, or any fragment thereof, thatdoes not include the cytoplasmic and/or transmembrane regions. Thecell-surface and cytoplasmic recombinant B7-related polypeptides can beisolated following cell lysis and extraction of cellular proteins, whilethe secreted recombinant B7-related polypeptides can be isolated fromthe cell growth media by standard techniques (see I. M. Rosenberg, Ed.(1996) Protein Analysis and Purification: Benchtop Techniques,Birkhauser, Boston, Cambridge, Mass.).

Antibody-based methods can used to purify natural or recombinantlyproduced B7-related polypeptides or peptides. Antibodies that recognizethese polypeptides, or peptides derived therefrom, can be produced andisolated using methods known and practiced in the art (see below).B7-related polypeptides or peptides can then be purified from a crudelysate by chromatography on antibody-conjugated solid-phase matrices(see E. Harlow and D. Lane (1999) Using Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Otherpurification methods known and used in the art may also be employed.

It is noted that transfected host cells that express B7-related factors(e.g., BSL1, BSL2, and/or BSL3) or portions thereof on the surface ofthe cell are within the scope of this invention. For example, a tumorcell such as a sarcoma, melanoma, leukemia, lymphoma, carcinoma, orneuroblastoma can be transfected with an expression vector directing theexpression of at least one B7-related factor on the surface of the tumorcell. Such transfected tumor cells can be used to treat tumor immunityas described in detail herein.

B7-Related Polypeptides

A further aspect of the present invention pertains to isolatedB7-related polypeptides. The present invention encompasses the BSL1(e.g., SEQ ID NO:2), BSL2 (e.g., SEQ ID NO:7, 11, or 13), or BSL3 (e.g.,SEQ ID NO:15) polypeptides, and fragments and functional equivalentsthereof. Such polypeptides can comprise at least 5, 12, 20, 30, 50, 90,100, 170, 200, 210, 300, or 500 contiguous amino acid residues.Preferred are polypeptides that share moderate homology with BSL1 (e.g.,SEQ ID NO:2), BSL2 (e.g., SEQ ID NO:7, 11, or 13), or BSL3 (e.g., SEQ IDNO:15) polypeptides. More preferred are polypeptides that sharesubstantial homology with BSL1 (e.g., SEQ ID NO:2), BSL2 (e.g., SEQ IDNO:7, 11, or 13), or BSL3 (e.g., SEQ ID NO:15).

The term “functional equivalent” is intended to include proteins whichdiffer in amino acid sequence from a given B7-related polypeptide, suchas sequence of BSL1 (e.g., SEQ ID NO:2), BSL2 (e.g., SEQ ID NO:7, 11, or13), or BSL3 (e.g., SEQ ID NO:15) polypeptide, but where suchdifferences result in a modified protein which performs at least onecharacteristic function of the B7-related polypeptide (e.g.,ligand-binding, antigenic, intra- or intercellular activity). Forexample, a functional equivalent of a BSL1, BSL2, or BSL3 polypeptidemay have a modification such as a substitution, addition or deletion ofan amino acid residue which is not directly involved in the function ofthis polypeptide (i.e., the ability of these polypeptides toco-stimulate T-cell proliferation). In addition, non-naturally occurringanalogs of B7-related polypeptides capable of binding CD28/CTLA-4 and/orCD28-/CTLA-4-related ligand(s) are considered functional equivalents.Various modifications of the B7-related polypeptides to producefunctional equivalents of these polypeptides are described in detailherein.

As described herein below, the BSL4-4616811 (BSL2vcvc) polypeptide wasdetermined to comprise a signal sequence from amino acid 1 to amino acid28 of SEQ ID NO:7 (FIG. 3B), according to the SPScan computer algorithm(Genetics Computer Group suite of programs). The site of signal sequencecleavage was confirmed by N-terminal sequencing of the BSL4-4616811polypeptide. Based on this data, the mature BSL4-4616811 (BSL2vcvc)polypeptide sequence includes amino acid 29 to amino acid 534 of SEQ IDNO:7 (FIG. 3B). As used herein a “mature sequence” is a polypeptidesequence that does not contain the signal sequence.

Accordingly, one embodiment of the present invention encompasses apolypeptide that lacks the signal sequence, but includes the remainingsequence of BSL2-4616811 (BSL2vcvc) polypeptide (i.e., the maturesequence of BSL2-4616811). Specifically, the invention encompasses apolypeptide comprising amino acids 29 through 534 of SEQ ID NO:7. Theinvention also encompasses a polynucleotide comprising nucleotides 85through 1602 of SEQ ID NO:131, as well as a polypeptide comprisingnucleotides 205 through 1722 of SEQ ID NO:6. Also encompassed arerecombinant vectors comprising these polynucleotides, and host cellscomprising these vectors.

Another embodiment of the present invention encompasses a polypeptidethat lacks the signal sequence, but includes the remaining sequence ofBSL2-L165-35b (BSL2v1c2) polypeptide, i.e., the mature sequence ofBSL2-L165-35b. Specifically, the invention encompasses a polypeptidecomprising amino acids 29 through 316 of SEQ ID NO:13. The inventionalso encompasses a polynucleotide comprising nucleotides 85 through 948of SEQ. ID NO:12. Also encompassed are recombinant vectors comprisingthese polynucleotides, and host cells comprising these vectors.

Yet another embodiment of the present invention encompasses apolypeptide that lacks the signal sequence, but includes the remainingsequence of BSL2-L165-21 (BSL2v2c2) polypeptide, i.e., the maturesequence of BSL2-L165-21. Specifically, the invention encompasses apolypeptide comprising amino acids 29 through 316 of SEQ ID NO:11. Theinvention also encompasses a polynucleotide comprising nucleotides 85through 948 of SEQ ID NO:10. Also encompassed are recombinant vectorscomprising these polynucleotides, and host cells comprising thesevectors.

It is also possible that under certain conditions the BSL2 signalsequence cleavage site may vary. The invention therefore encompassespolypeptides that add or subtract 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 more contiguous amino acids from theN-terminus of the polypeptides described in the three proceedingparagraphs. Polynucleotides encoding these polypeptides are alsoencompassed by the invention, as well as vectors and host cellscomprising these polynucleotides.

It is possible to modify the structure of a B7-related polypeptide forsuch purposes as increasing solubility, enhancing therapeutic orprophylactic efficacy (reactivity), or stability (e.g., shelf life exvivo and resistance to proteolytic degradation in vivo). Such modifiedproteins are considered functional equivalents of the B7-relatedpolypeptides as defined herein. Preferably, the B7-related polypeptidesare modified so that they retain the ability to modulate (e.g.,co-stimulate or inhibit) T-cell proliferation. Those residues shown tobe essential to interact with the CD28/CTLA-4 or CD28-/CTLA-4-relatedligands on T-cells can be modified by replacing the essential amino acidwith another, preferably similar amino acid residue (a conservativesubstitution) whose presence is shown to enhance, diminish, but noteliminate, or not effect receptor interaction. In addition, those aminoacid residues that are not essential for receptor interaction can bemodified by being replaced by another amino acid whose incorporation mayenhance, diminish, or not effect reactivity. For example, a B7-relatedpolypeptide can be modified by substitution of cysteine residues withother amino acids, such as alanine, serine, threonine, leucine, orglutamic acid, to prevent dimerization via disulfide linkages. Inaddition, the amino acid side chains of a B7-related polypeptide of theinvention can be chemically modified. Also, a B7-related polypeptide canbe modified by cyclization of the amino acid sequence.

In order to enhance stability and/or reactivity, the B7-relatedpolypeptides can be altered to incorporate one or more polymorphisms inthe amino acid sequence. Additionally, D-amino acids, non-natural aminoacids, or non-amino acid analogs can be substituted or added to producea modified polypeptide. Furthermore, the B7-related polypeptidesdisclosed herein can be modified using polyethylene glycol (PEG)according to known methods (Wie et al., supra) to produce a proteinconjugated with PEG. In addition, PEG can be added during chemicalsynthesis of the protein. Other possible modifications includereduction/alkylation (Tarr (1986) Methods of ProteinMicrocharacterization, J. E. Silver, Ed., Humana Press, Clifton, N.J.,pp. 155-194); acylation (Tarr, supra); chemical coupling to anappropriate carrier (Mishell and Shiigi, Eds. (1980) Selected Methods inCellular Immunology, WH Freeman, San Francisco, Calif.; U.S. Pat. No.4,939,239; or mild formalin treatment (Marsh (1971) Int. Arch. ofAllergy and Appl. Immunol. 41:199-215) of the B7-related polypeptide.

Modified polypeptides can have conservative changes, wherein asubstituted amino acid has similar structural or chemical properties,e.g., replacement of leucine with isoleucine. More infrequently, amodified polypeptide can have non-conservative changes, e.g.,substitution of a glycine with a tryptophan. Guidance in determiningwhich amino acid residues can be substituted, inserted, or deletedwithout abolishing biological or immunological activity can be foundusing computer programs well known in the art, for example, DNASTARsoftware (DNASTAR, Inc., Madison, Wis.)

As non-limiting examples, conservative substitutions in the B7-relatedamino acid sequence can be made in accordance with the following table.Original Conservative Residue Substitution(s) Ala Ser Arg Lys Asn Gln,His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu, ValLeu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr Ser ThrThr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function or immunogenicity can be made byselecting substitutions that are less conservative than those shown inthe table, above. For example, non-conservative substitutions can bemade which more significantly affect the structure of the polypeptide inthe area of the alteration, for example, the alpha-helical, orbeta-sheet structure; the charge or hydrophobicity of the molecule atthe target site; or the bulk of the side chain. The substitutions whichgenerally are expected to produce the greatest changes in thepolypeptide's properties are those where 1) a hydrophilic residue, e.g.,seryl or threonyl, is substituted for (or by) a hydrophobic residue,e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; 2) a cysteineor proline is substituted for (or by) any other residue; 3) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or 4) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) a residue that does not have aside chain, e.g., glycine.

Preferred polypeptide embodiments further include an isolatedpolypeptide comprising an amino acid sequence sharing at least 60, 70,80, 85, 90, 95, 97, 98, 99, 99.5 or 100% identity with an amino acidsequence of BSL1 (e.g., SEQ ID NO:2), BSL2 (e.g., SEQ ID NO:7, 11, or13), or BSL3 (e.g., SEQ ID NO:15). This polypeptide sequence may beidentical to the sequence of BSL1 (e.g., SEQ ID NO:2), BSL2 (e.g., SEQID NO:7, 11, or 13), or BSL3 (e.g., SEQ ID NO:15), or may include up toa certain integer number of amino acid alterations as compared to thereference sequence

Percent sequence identity can be calculated using computer programs ordirect sequence comparison. Preferred computer program methods todetermine identity between two sequences include, but are not limitedto, the GCG program package, FASTA, BLASTP, and TBLASTN (see, e.g., D.W. Mount, 2001, Bioinformatics: Sequence and Genome Analysis, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The BLASTPand TBLASTN programs are publicly available from NCBI and other sources.The well-known Smith Waterman algorithm may also be used to determineidentity.

Exemplary parameters for amino acid sequence comparison include thefollowing: 1) algorithm from Needleman and Wunsch (1970) J. Mol. Biol.48:443-453; 2) BLOSSUM62 comparison matrix from Hentikoff and Hentikoff(1992) Proc. Natl. Acad. Sci. USA 89:10915-10919; 3) gap penalty=12; and4) gap length penalty=4. A program useful with these parameters ispublicly available as the “gap” program (Genetics Computer Group,Madison, Wis.). The aforementioned parameters are the default parametersfor polypeptide comparisons (with no penalty for end gaps).

Alternatively, polypeptide sequence identity can be calculated using thefollowing equation: % identity=(the number of identicalresidues)/(alignment length in amino acid residues)*100. For thiscalculation, alignment length includes internal gaps but does notinclude terminal gaps.

In accordance with the present invention, polypeptide sequences may beidentical to the sequence of BSL1 (e.g., SEQ ID NO:2), BSL2 (e.g., SEQID NO:7, 11, or 13), or BSL3 (e.g., SEQ ID NO:15), or may include up toa certain integer number of amino acid alterations. Polypeptidealterations are selected from the group consisting of at least one aminoacid deletion, substitution, including conservative and non-conservativesubstitution, or insertion. Alterations may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence. In specificembodiments, polypeptide variants may be encoded by BSL1, BSL2, or BSL3nucleic acids comprising single nucleotide polymorphisms and/oralternate splice variants. Polypeptides may also be modified by, forexample, phosphorylation, sulfation, or acylation. They may also bemodified with a label capable of providing a detectable signal, eitherdirectly or indirectly, including, but not limited to, radioisotopes andfluorescent compounds.

In addition, the B7-related polypeptides of the invention can be fusedto heterologous peptide or polypeptide sequences to create fusionproteins such as BSL1-Ig (e.g., SEQ ID NO:5); BSL2-4616811-Ig (e.g., SEQID NO:9), BSL2-L165-35b-Ig (e.g., SEQ ID NO:133), BSL2-L165-21-Ig (e.g.,SEQ ID NO:135), and BSL3-Ig (e.g., SEQ ID NO: 17) as described in detailherein. In accordance with the experiments of the invention, the BSL2sequence of BSL2-4616811-Ig (BSL2vcvc-Ig) polypeptide was determined tocomprise a signal sequence from about amino acid 1 to about amino acid28 of SEQ ID NO:9 (FIG. 4B). The signal sequence cleavage site wasdetermined using the SPScan computer algorithm (Genetics Computer Groupsuite of programs), and was confirmed by N-terminal sequencing. Based onthis data, the mature BSL2-4616811-Ig (BSL2vcvc-Ig) polypeptide sequenceextends from amino acids 29 through 698 of SEQ ID NO:9 (FIG. 4B).

Accordingly, one embodiment of the present invention encompasses aBSL2-4616811-Ig (BSL2vcvc-Ig) polypeptide that includes amino acids 1through 698 of SEQ ID NO:9. The invention also encompasses aBSL2-4616811-Ig (BSL2vcvc-Ig) polypeptide that lacks the initiatingmethionine, but includes amino acids 2 through 698 of SEQ ID NO:9. Theinvention further encompasses a polypeptide that lacks the signalsequence, but includes the remaining sequence of BSL2-4616811-Ig(BSL2vcvc-Ig) polypeptide, i.e., the mature sequence of BSL2-4616811-Ig.Specifically, the invention encompasses a polypeptide comprising aminoacids 29 through 698 of SEQ ID NO:9. The invention also encompassesBSL2-4616811-Ig (BSL2vcvc-Ig) polynucleotides comprising nucleotides 1through 2094, nucleotides 4 through 2094, or nucleotides 85 through 2094of SEQ ID NO:8. Also encompassed are recombinant vectors comprisingthese polynucleotides, and host cells comprising these vectors.

Another embodiment of the present invention encompasses aBSL2-L165-35b-Ig (BSL2v1c2-Ig) polypeptide that includes amino acids 1through 480 of SEQ ID NO:133. The invention also encompasses aBSL2-L165-35b-Ig (BSL2v1c2-Ig) polypeptide that lacks the initiatingmethionine, but includes amino acids 2 through 480 of SEQ ID NO:133. Theinvention further encompasses a polypeptide that lacks the signalsequence, but includes the remaining sequence of BSL2-L165-35b-Ig(BSL2v1c2-Ig) polypeptide, i.e., the mature sequence ofBSL2-L165-35b-Ig. Specifically, the invention encompasses a polypeptidecomprising amino acids 29 through 480 of SEQ ID NO:133. The inventionalso encompasses BSL2-L165-35b-Ig (BSL2v1c2-Ig) polynucleotidescomprising nucleotides 1 through 1440, nucleotides 4 through 1440, ornucleotides 85 through 1440 of SEQ ID NO:132. Also encompassed arerecombinant vectors comprising these polynucleotides, and host cellscomprising these vectors.

Yet another embodiment of the present invention encompasses aBSL2-L-165-21-Ig (BSL2v2c2-Ig) polypeptide that includes amino acids 1through 480 of SEQ ID NO:135. The invention also encompasses aBSL2-L165-21-Ig (BSL2v2c2-Ig) polypeptide that lacks the initiatingmethionine, but includes amino acids 2 through 480 of SEQ ID NO:135. Theinvention further encompasses a polypeptide that lacks the signalsequence, but includes the remaining sequence of BSL2-L165-21-Ig(BSL2v2c2-Ig) polypeptide, i.e., the mature sequence of BSL2-L165-21-Ig.Specifically, the invention encompasses a BSL2-L165-21-Ig (BSL2v2c2-Ig)polypeptide comprising amino acids 29 through 480 of SEQ ID NO:135. Theinvention also encompasses BSL2-L165-21-Ig (BSL2v2c2-Ig) polynucleotidescomprising nucleotides 1 through 1440, nucleotides 4 through 1440, ornucleotides 85 through 1440 of SEQ ID NO:134. Also encompassed arerecombinant vectors comprising these polynucleotides, and host cellscomprising these vectors.

It is also possible that under certain conditions the BSL2 signalsequence cleavage site may vary. The invention therefore encompassespolypeptides that add or subtract 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 more contiguous amino acids from theN-terminus of the polypeptides described in the three proceedingparagraphs. Polynucleotides encoding these polypeptides are alsoencompassed by the invention, as well as vectors and host cellscomprising these polynucleotides.

The invention also relates to isolated, synthesized and/or recombinantportions or fragments of a BSL1 (e.g., SEQ ID NO:2), BSL2 (e.g., SEQ IDNO:7, 11, or 13), or BSL3 (e.g., SEQ ID NO:15) protein or polypeptide asdescribed herein. Polypeptide fragments (i.e., peptides) can be madewhich have full or partial function on their own, or which when mixedtogether (though fully, partially, or nonfunctional alone),spontaneously assemble with one or more other polypeptides toreconstitute a functional protein having at least one functionalcharacteristic of a BSL1, BSL2, or BSL3 protein of this invention. Inaddition, B7-related polypeptide fragments may comprise, for example,one or more domains of the polypeptide (e.g., the transmembrane orextracellular domain) disclosed herein.

The polypeptides of the present invention, includingfunction-conservative variants, may be isolated from wild-type or mutantcells (e.g., human cells or cell lines), from heterologous organisms orcells (e.g., bacteria, yeast, insect, plant, and mammalian cells), orfrom cell-free translation systems (e.g., wheat germ, microsomalmembrane, or bacterial extracts) in which a protein-coding sequence hasbeen introduced and expressed. Furthermore, the polypeptides may be partof recombinant fusion proteins. The polypeptides can also,advantageously, be made by synthetic chemistry. Polypeptides may bechemically synthesized by commercially available automated procedures,including, without limitation, exclusive solid phase synthesis, partialsolid phase methods, fragment condensation or classical solutionsynthesis. Both the naturally occurring and recombinant forms of thepolypeptides of the invention can advantageously be used to screencompounds for binding activity. The polypeptides of the invention alsofind use as therapeutic agents as well as antigenic components toprepare antibodies as described in detail herein.

Antibodies to B7-Related Polypeptides

Antibodies directed against the B7-related polypeptides of the presentinvention, e.g., BSL1 (e.g., SEQ ID NO:2), BSL2 (e.g., SEQ ID NO:7, 11,or 13), or BSL3 (e.g., SEQ ID NO:15), or antigenic or immunogenicepitopes thereof, can be, for example, polyclonal or monoclonalantibodies. The present invention also includes chimeric, single chain,and humanized antibodies, as well as Fab, F(ab′)₂, or Fv fragments, orthe product of an Fab expression library. Various procedures known inthe art may be used for the production of such antibodies and antibodyfragments.

Antibodies generated against the polypeptides or peptides correspondingto one or more of the B7-related sequences of the present invention canbe obtained by direct injection of the polypeptides or peptides into ananimal, or by administering the polypeptides or peptides to an animal,preferably a nonhuman animal. The antibodies so obtained will then bindto the polypeptides or peptides. In this manner, even a sequenceencoding only a fragment of a polypeptide can be used to generateantibodies binding to the whole native polypeptide. Such antibodies canbe used, for example, to isolate the polypeptide from tissue expressingthat polypeptide.

For the preparation of monoclonal antibodies, any technique thatprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler and Milstein(1975) Nature, 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al. (1983) Immunol. Today, 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole etal. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96). Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic polypeptide products of this invention.Also, transgenic mice may be used to express humanized antibodies toimmunogenic polypeptide products of this invention.

The present invention encompasses polypeptides comprising, oralternatively, consisting of, an epitope of the polypeptide having anamino acid sequence of one or more of the BSL1, BSL2, or BSL3 amino acidsequences as set forth in FIGS. 1-6. The present invention furtherencompasses polynucleotide sequences encoding an epitope of apolypeptide sequence of BSL1, BSL2, or BSL3 of the invention. Typically,BSL1, BSL2, or BSL3 epitopes comprise hydrophilic regions of thecorresponding polypeptides (e.g., SEQ ID NO:2, SEQ ID NO:7, 11, or 13,or SEQ ID NO:15). Hydrophilic regions can be determined by any methodknown in the art, for example, Kyte-Doolittle Hydrophilicity Plots(e.g., using the program bundle from Genetics Computer Group). Inaddition, the antigenic index can be determined directly using theJameson-Wolf method (e.g., using the program bundle from GeneticsComputer Group).

Non-limiting examples of BSL2-4616811 (BSL2vcvc) sequences which may beused as epitopes include sequences comprising amino acids 68 through109; amino acids 148 through 186; amino acids 284 through 326; or aminoacids 361 through 407 of SEQ ID NO:7. This invention also encompassespolynucleotides encoding these epitopes, and vectors and host cellscomprising these polynucleotides. For example, such polynucleotides maycomprise nucleotides 202 through 327; nucleotides 442 through 558;nucleotides 850 through 978; or nucleotides 1081 through 1221 of SEQ IDNO:131. Similarly, these polynucleotides may comprise nucleotides 322through 447; nucleotides 562 through 678; nucleotides 970 through 1098;or nucleotides 1201 through 1341 of SEQ ID NO:6.

In preferred embodiments, the following immunogenic and/or antigenicepitopes are encompassed by the present invention: eptitopes comprisingfrom about amino acid 68 to about amino acid 74, from about amino acid75 to about amino acid 81, from about amino acid 82 to about amino acid88, from about amino acid 89 to about amino acid 95, from about aminoacid 96 to about amino acid 102, from about amino acid 103 to aboutamino acid 109, from about amino acid 148 to about amino acid 154, fromabout amino acid 155 to about amino acid 161, from about amino acid 162to about amino acid 168, from about amino acid 169 to about amino acid175, from about amino acid 176 to about amino acid 182, from about aminoacid 183 to about amino acid 186, from about amino acid 284 to aboutamino acid 290, from about amino acid 291 to about amino acid 297, fromabout amino acid 298 to about amino acid 304, from about amino acid 305to about amino acid 311, from about amino acid 312 to about amino acid318, from about amino acid 319 to about amino acid 326, from about aminoacid 361 to about amino acid 367, from about amino acid 368 to aboutamino acid 374, from about amino acid 375 to about amino acid 381, fromabout amino acid 387 to about amino acid 393, from about amino acid 394to about amino acid 400, and/or from about amino acid 401 to about aminoacid 407 of SEQ ID NO:7. In this context, the term “about” should beconstrued to mean 1, 2, 3, 4, or 5 more amino acids in either the N- orC-terminal direction of the above referenced epitopes. Polynucleotidesencoding these polypeptides are also provided, as well as vectors andhost cells comprising these polynucleotides.

The term “epitopes” as used herein, refers to portions of a polypeptide(e.g., peptides) having antigenic or immunogenic activity in an animal,preferably a mammal, and most preferably a human. In a preferredembodiment, the present invention encompasses a polypeptide comprisingan epitope, as well as the polynucleotide encoding this polypeptide. An“immunogenic epitope” as used herein, refers to a portion of a proteinthat elicits an antibody response in an animal, as determined by anymethod known in the art, for example, by the methods for generatingantibodies described infra. (See, for example, Geysen et al. (1983)Proc. Natl. Acad. Sci. USA, 81:3998-4002). The term “antigenic epitope”as used herein refers to a portion of a protein to which an antibody canimmunospecifically bind to its antigen as determined by any method wellknown in the art, for example, by the immunoassays described herein.Immunospecific binding excludes non-specific binding, but does notnecessarily exclude cross-reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic. Either the full-lengthprotein or an antigenic peptide fragment can be used. Antibodies arepreferably prepared from these regions or from discrete fragments inregions of the BSL1, BSL2, or BSL3 nucleic acid and amino acid sequencescomprising an epitope.

Moreover, antibodies can also be prepared from any region of thepolypeptides and peptides of the B7-related sequences as describedherein. A preferred fragment generates the production of an antibodythat diminishes or completely prevents interaction with a bindingpartner. In addition, antibodies can be developed against an entire BSL1(e.g., SEQ ID NO:2), BSL2 (e.g., SEQ ID NO:7, 11, or 13), or BSL3 (e.g.,SEQ ID NO:15) polypeptide or portions of the polypeptide, for example, acarboxy-terminal domain, an amino-terminal extracellular domain, anentire transmembrane domain, specific transmembrane segments, or anyportions of these regions. Antibodies can also be developed againstspecific functional sites, such as the site of binding, or sites thatare glycosylated, phosphorylated, myristylated, or amidated, forexample. Also useful for antibody production are variable/constant (vc)domains of the B7-related polypeptides, e.g., the v1c2, v2c2, orv1c1v2c2 domains of BSL2 (see below). Polypeptide or peptide fragmentsthat function as epitopes may be produced by any conventional means.(See, e.g., Houghten (1985) Proc. Natl. Acad. Sci. USA, 82:5131-5135;and as described in U.S. Pat. No. 4,631,211).

In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 20, at least 25, atleast 30, at least 40, at least 50, and, most preferably, between about15 to about 30 contiguous amino acids. Preferred polypeptides comprisingimmunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 contiguous aminoacid residues in length. Additional non-exclusive preferred antigenicepitopes include the antigenic epitopes disclosed herein, as well asportions thereof, as well as any combination of two, three, four, fiveor more of these antigenic epitopes. Antigenic epitopes are useful, forexample, to raise antibodies, including monoclonal antibodies thatspecifically bind the epitope. In addition, antigenic epitopes can beused as the target molecules in immunoassays. (See, for instance, Wilsonet al. (1984) Cell, 37:767-778; and Sutcliffe et al. (1983) Science,219:660-666). Such fragments as described herein are not to beconstrued, however, as encompassing any fragments that may be disclosedprior to the invention.

Similarly, immunogenic epitopes can be used, for example, to induceantibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.(1985) Proc. Natl. Acad. Sci. USA, 82:910-914; and Bittle et al. (1985)J. Gen. Virol., 66:2347-2354). Preferred immunogenic epitopes includethe immunogenic epitopes disclosed herein, as well as any combination oftwo, three, four, five or more of these immunogenic epitopes.

B7-related polypeptides of the invention comprising one or moreimmunogenic epitopes that elicit an antibody response can beintroduction together with a carrier protein, such as an albumin, to ananimal system (such as rabbit or mouse). Alternatively, if thepolypeptide is of sufficient length (e.g., at least about 25 contiguousamino acids), the polypeptide can be presented without a carrier.However, immunogenic epitopes comprising as few as 5 to 10 amino acidshave been shown to be sufficient to raise antibodies capable of bindingto, at the very least, linear epitopes in a denatured polypeptide (e.g.,in Western blotting).

Epitope-bearing polypeptides of the present invention may be used toinduce antibodies according to methods well known in the art including,but not limited to, in vivo immunization, in vitro immunization, andphage display methods. See, e.g., Sutcliffe et al., supra; Wilson etal., supra; and Bittle et al., supra). If in vivo immunization is used,animals can be immunized with free peptide; however, the anti-peptideantibody titer may be boosted by coupling the peptide to amacromolecular carrier, such as keyhole limpet hemacyanin (KLH), ortetanus toxoid (TT). For instance, peptides containing cysteine residuescan be coupled to a carrier using a linker such asmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent, such asglutaraldehyde.

Epitope bearing peptides of the invention may also be synthesized asmultiple antigen peptides (MAPs), first described by J. P. Tam et al.(1995) Biomed. Pept, Proteins, Nucleic Acids, 199, 1(3):123-32; andCalvo, et al. (1993) J. Immunol., 150(4):1403-12), which are herebyincorporated by reference in their entirety herein. MAPs containmultiple copies of a specific peptide attached to a non-immunogeniclysine core. MAP peptides usually contain four or eight copies of thepeptide, which are often referred to as MAP4 or MAP8 peptides. By way ofnon-limiting example, MAPs can be synthesized onto a lysine core matrixattached to a polyethylene glycol-polystyrene (PEG-PS) support. Thepeptide of interest is synthesized onto the lysine residues using9-fluorenylmethoxycarbonyl (Fmoc) chemistry. For example, AppliedBiosystems (Foster City, Calif.) offers commercially available MAPresins, such as, for example, the Fmoc Resin 4 Branch and the Fmoc Resin8 Branch, which can be used to synthesize MAPs. Cleavage of MAPs fromthe resin is performed with standard trifloroacetic acid (TFA)-basedcocktails known in the art. Purification of MAPs, except for desalting,is not generally necessary. MAP peptides can be used in immunizingvaccines which elicit antibodies that recognize both the MAP and thenative protein from which the peptide was derived.

Epitope-bearing peptides of the invention can also be incorporated intoa coat protein of a virus, which can then be used as an immunogen or avaccine with which to immunize animals, including humans, in orderstimulate the production of anti-epitope antibodies. For example, the V3loop of the gp120 glycoprotein of the human immunodeficiency virus type1 (HIV-1) has been engineered to be expressed on the surface ofrhinovirus. Immunization with rhinovirus displaying the V3 loop peptideyielded apparently effective mimics of the HIV-1 immunogens (as measuredby their ability to be neutralized by anti-HIV-1 antibodies as well asby their ability to elicit the production of antibodies capable ofneutralizing HIV-1 in cell culture). This techniques of using engineeredviral particles as immunogens is described in more detail in Smith etal. (1997) Behring Inst Mitt Feb, 98:229-39; Smith et al. (1998) J.Virol. 72:651-659; and Zhang et al. (1999) Biol. Chem. 380:365-74),which are hereby incorporated by reference herein in their entireties.

Epitope bearing polypeptides of the invention can be modified, forexample, by the addition of amino acids at the amino- and/orcarboxy-terminus of the peptide. Such modifications are performed, forexample, to alter the conformation of the epitope bearing polypeptidesuch that the epitope will have a conformation more closely related tothe structure of the epitope in the native protein. An example of amodified epitope-bearing polypeptide of the invention is a polypeptidein which one or more cysteine residues have been added to thepolypeptide to allow for the formation of a disulfide bond between twocysteines, thus resulting in a stable loop structure of theepitope-bearing polypeptide under non-reducing conditions. Disulfidebonds can form between a cysteine residue added to the polypeptide and acysteine residue of the naturally-occurring epitope, or between twocysteines which have both been added to the naturally-occurringepitope-bearing polypeptide.

In addition, it is possible to modify one or more amino acid residues ofthe naturally-occurring epitope-bearing polypeptide by substitution withcysteines to promote the formation of disulfide bonded loop structures.Cyclic thioether molecules of synthetic peptides can be routinelygenerated using techniques known in the art, e.g., as described in PCTpublication WO 97/46251, incorporated in its entirety by referenceherein. Other modifications of epitope-bearing polypeptides contemplatedby this invention include biotinylation.

For the production of antibodies in vivo, host animals, such as rabbits,rats, mice, sheep, or goats, are immunized with either free orcarrier-coupled peptides or MAP peptides, for example, byintraperitoneal and/or intradermal injection. Injection material istypically an emulsion containing about 100 μg of peptide or carrierprotein and Freund's adjuvant, or any other adjuvant known forstimulating an immune response. Several booster injections may beneeded, for instance, at intervals of about two weeks, to provide auseful titer of anti-peptide antibody which can be detected, forexample, by ELISA assay using free peptide adsorbed to a solid surface.The titer of anti-peptide antibodies in serum from an immunized animalcan be increased by selection of anti-peptide antibodies, e.g., byadsorption of the peptide onto a solid support and elution of theselected antibodies according to methods well known in the art.

As one having skill in the art will appreciate, and as discussed above,the B7-related polypeptides of the present invention, which comprise animmunogenic or antigenic epitope, can be fused to other polypeptidesequences. For example, the polypeptides of the present invention can befused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgD,or IgM), or portions thereof, e.g., CH1, CH2, CH3, or any combinationthereof, and portions thereof, or with albumin (including, but notlimited to, recombinant human albumin, or fragments or variants thereof(see, e.g., U.S. Pat. No. 5,876,969; EP Patent No. 0 413 622; and U.S.Pat. No. 5,766,883, incorporated by reference in their entirety herein),thereby resulting in chimeric polypeptides. Such fusion proteins mayfacilitate purification and may increase half-life in vivo. This hasbeen shown for chimeric proteins containing the first two domains of thehuman CD4-polypeptide and various domains of the constant regions of theheavy or light chains of mammalian immunoglobulins. See, e.g.,Traunecker et al. (1988) Nature 331:84-86).

Enhanced delivery of an antigen across the epithelial barrier to theimmune system has been demonstrated for antigens (e.g., insulin)conjugated to an FcRn binding partner, such as IgG or Fc fragments (see,e.g., PCT Publications WO 96/22024 and WO 99/04813). IgG fusion proteinsthat have a disulfide-linked dimeric structure due to the IgG portiondisulfide bonds have also been found to be more efficient in binding andneutralizing other molecules than are monomeric polypeptides, orfragments thereof, alone. See, e.g., Fountoulakis et al. (1995) J.Biochem. 270:3958-3964).

Nucleic acids encoding epitopes can also be recombined with a gene ofinterest as an epitope tag (e.g., the hemagglutinin (“HA”) tag or flagtag) to aid in detection and purification of the expressed polypeptide.For example, a system for the ready purification of non-denatured fusionproteins expressed in human cell lines has been described (Janknecht etal. (1991) Proc. Natl. Acad. Sci. USA, 88:8972-897). In this system, thegene of interest is subcloned into a vaccinia recombination plasmid suchthat the open reading frame of the gene is translationally fused to anamino-terminal tag having six histidine residues. The tag serves as amatrix binding domain for the fusion protein. Extracts from cellsinfected with the recombinant vaccinia virus are loaded onto a Ni²⁺nitriloacetic acid-agarose column and histidine-tagged proteins areselectively eluted with imidazole-containing buffers.

Additional fusion proteins of the invention can be generated byemploying the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”). DNA shuffling can be employed to modulate the activities ofpolypeptides of the invention, such methods can be used to generatepolypeptides with altered activity, as well as agonists and antagonistsof the polypeptides. See, generally, U.S. Pat. Nos. 5,605,793;5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al. (1997)Curr. Opin. Biotechnol. 8:724-33; Harayama (1998) Trends Biotechnol.16(2):76-82; Hansson, et al. (1999) J. Mol. Biol. 287:265-76; andLorenzo and Blasco (1998) Biotechniques, 24(2):308-313, the contents ofeach of which are hereby incorporated by reference in its entirety).

In an embodiment of the invention, alteration of polynucleotidescorresponding to one or more of the B7-related polynucleotide sequencesas set forth in FIGS. 1-6, and the polypeptides encoded by thesepolynucleotides, can be achieved by DNA shuffling. DNA shufflinginvolves the assembly of two or more DNA segments by homologous orsite-specific recombination to generate variation in the polynucleotidesequence. In another embodiment, polynucleotides of the invention, ortheir encoded polypeptides, may be altered by being subjected to randommutagenesis by error-prone PCR, random nucleotide insertion, or othermethods, prior to recombination. In another embodiment, one or morecomponents, motifs, sections, parts, domains, fragments, etc., of apolynucleotide encoding a polypeptide of this invention may berecombined with one or more components, motifs, sections, parts,domains, fragments, etc. of one or more heterologous molecules.

Another aspect of the present invention relates to antibodies and T-cellantigen receptors (TCRs), which immunospecifically bind to apolypeptide, polypeptide fragment, or variant one or more of the BSL1,BSL2, or BSL3 amino acid sequences as set forth in FIGS. 1-6, and/or anepitope thereof, of the present invention (as determined by immunoassayswell known in the art for assaying specific antibody-antigen binding).The basic antibody structural unit of an antibody or immunoglobulin isknown to comprise a tetramer. Each tetramer is composed of two identicalpairs of polypeptide chains, each pair having one “light” (about 25 kDa)and one “heavy” chain (about 50-70 kDa). The amino terminal portion ofeach chain includes a variable region of about 100 to 110 or more aminoacids; the variable region is primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region that is primarily responsible for immunoglobulineffector function. Immunoglobulin light chains, including human lightchains, are of the kappa and lambda types. Immunoglobulin heavy chainisotypes include IgM, IgD, IgG, IgA, and IgE. (See, generally, W. Paul,Ed., (1989) Fundamental Immunology, Ch. 7, 2nd ed., Raven Press, N.Y.,incorporated herein by reference in its entirety). The variable regionsof each light/heavy chain pair form the antibody or immunoglobulinbinding site. Thus, for example, an intact IgG antibody has two bindingsites. Except in bifunctional or bispecific antibodies, the two bindingsites are the same.

The chains of an immunoglobulin molecule exhibit the same generalstructure of relatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. The CDRs of the heavy and the light chains of each pair arealigned by the framework regions, thus enabling binding to a specificepitope. From N-terminus to C-terminus, both the light and heavy chainscomprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)); Chothiaand Lesk (1987) J. Mol. Biol. 196:901-917; or Chothia et al. (1989)Nature, 342:878-883.

A bispecific or bifunctional antibody is an artificial hybrid antibodyhaving two different heavy/light chain pairs and two different bindingsites. Bispecific antibodies can be produced by a variety of methods,including fusion of hybridomas or linking of Fab′ fragments. (See, e.g.,Songsivilai & Lachmann (1990) Clin. Exp. Immunol. 79:315-321; Kostelnyet al. (1992) J. Immunol. 148:1547 1553). In addition, bispecificantibodies can be formed as “diabodies” (See, Holliger et al. (1993)Proc. Natl. Acad. Sci. USA, 90:6444-6448), or “Janusins” (See,Traunecker et al. (1991) EMBO J., 10:3655-3659 and Traunecker et al.(1992) Int. J. Cancer Suppl. 7:51-52).

Antibodies of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-id antibodies to antibodies of theinvention), intracellularly made antibodies (i.e., intrabodies), andepitope-binding fragments of any of the above. The term “antibody”, asused herein, refers to immunoglobulin molecules and immunologicallyactive portions or fragments of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The immunoglobulin molecules of the invention can beof any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class or subclass(e.g., IgG1, IgG2, IgG3, IgG4, IgAI and IgA2) of immunoglobulinmolecule. In a preferred embodiment, the immunoglobulin is an IgG1isotype. In another preferred embodiment, the immunoglobulin is an IgG2isotype. In another preferred embodiment, the immunoglobulin is an IgG4isotype.

Immunoglobulins may have both a heavy and a light chain. An array ofIgG, IgE, IgM, IgD, IgA, and IgY heavy chains can be paired with a lightchain of the kappa or lambda types. Most preferably, the antibodies ofthe present invention are human antigen-binding antibodies and antibodyfragments and include, but are not limited to, Fab, Fab′ F(ab′) 2, Fd,single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs(sdFv) and fragments comprising either a V_(L) or V_(H) domain.Antigen-binding antibody fragments, including single-chain antibodies,can comprise the variable region(s) alone or in combination with theentirety or a portion of the following: hinge region, and CH1, CH2, andCH3 domains. Also included in connection with the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, and CH1, CH2, and CH3 domains. Theantibodies of the invention may be from any animal origin includingbirds and mammals. Preferably, the antibodies are of human, murine(e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel,horse, or chicken origin. As used herein, “human” antibodies includeantibodies having the amino acid sequence of a human immunoglobulin andinclude antibodies isolated from human immunoglobulin libraries or fromanimals transgenic for one or more human immunoglobulin and that do notexpress endogenous immunoglobulins, as described infra and, for example,in U.S. Pat. No. 5,939,598.

The antibodies of the present invention can be monospecific, bispecific,trispecific, or of greater multispecificity. Multispecific antibodiescan be specific for different epitopes of a polypeptide of the presentinvention, or can be specific for both a polypeptide of the presentinvention, and a heterologous epitope, such as a heterologouspolypeptide or solid support material. (See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al. (1991) J.Immunol. 147:60-69; U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648;5,573,920; 5,601,819; and Kostelny et al. (1992) J. Immunol.148:1547-1553).

Antibodies of the present invention can be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention that they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) can be specified, e.g., by N-terminal andC-terminal positions, by size in contiguous amino acid residues, or aspresented in the sequences defined in FIGS. 1-6, herein. Furtherincluded in accordance with the present invention are antibodies thatbind to polypeptides encoded by polynucleotides that hybridize to apolynucleotide of the present invention under stringent, or moderatelystringent, hybridization conditions as described herein.

The antibodies of the invention (including molecules comprising, oralternatively consisting of, antibody fragments or variants thereof) canbind immunospecifically to a polypeptide or polypeptide fragment orvariant human B7-related polypeptide as set forth in FIGS. 1-6 and/ormonkey B7-related polypeptide.

By way of non-limiting example, an antibody can be considered to bind toa first antigen preferentially if it binds to the first antigen with adissociation constant (Kd) that is less than the antibody's Kd for thesecond antigen. In another non-limiting embodiment, an antibody can beconsidered to bind to a first antigen preferentially if it binds to thefirst antigen with an affinity that is at least one order of magnitudeless than the antibody's association constant (Ka) for the secondantigen. In another non-limiting embodiment, an antibody can beconsidered to bind to a first antigen preferentially if it binds to thefirst antigen with an affinity that is at least two orders of magnitudeless than the antibody's Kd for the second antigen.

In another nonlimiting embodiment, an antibody may be considered to bindto a first antigen preferentially if it binds to the first antigen withan off rate (Koff) that is less than the antibody's Koff for the secondantigen. In another nonlimiting embodiment, an antibody can beconsidered to bind to a first antigen preferentially if it binds to thefirst antigen with an affinity that is at least one order of magnitudeless than the antibody's Koff for the second antigen. In anothernonlimiting embodiment, an antibody can be considered to bind to a firstantigen preferentially if it binds to the first antigen with an affinitythat is at least two orders of magnitude less than the antibody's Kofffor the second antigen.

Antibodies of the present invention can also be described or specifiedin terms of their binding affinity to a B7-related polypeptide of thepresent invention. Preferred binding affinities include those with adissociation constant or Kd of less than 5×10⁻² M, 1×10⁻² M, 5×10⁻³ M,1×10⁻³ M, 5×10⁻⁴ M, or 1×10⁻⁴ M. More preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10⁻⁵ M,1×10⁻⁵ M, 5×10⁻⁶ M, 1×10⁻⁶ M, 5×10⁻⁷ M, 1×10⁻⁷ M, 5×10⁻⁸ M, or 1×10⁻⁸ M.Even more preferred antibody binding affinities include those with adissociation constant or Kd of less than 5×10⁻⁹ M, 1×10⁻⁹ M, 5×10⁻¹⁰ M,1×10⁻¹⁰ M, 5×10⁻¹¹ M, 1×10⁻¹¹ M, 5×10⁻¹² M, 1×10⁻¹² M, 5×10⁻¹³ M,1×10⁻¹³ M, 5×10⁻¹⁴ M, 1×10⁻¹⁴ M, 5×10⁻¹⁵ M, or 1×10⁻¹⁵ M.

In specific embodiments, antibodies of the invention bind to B7-relatedpolypeptides of the invention, or fragments, or variants thereof, withan off rate (Koff) of less than or equal to about 5×10⁻² sec⁻¹, 1×10⁻²sec⁻¹, 5×10⁻³ sec⁻¹, or 1×10⁻³ sec⁻¹. More preferably, antibodies of theinvention bind to B7-related polypeptides of the invention or fragmentsor variants thereof with an off rate (Koff) of less than or equal toabout 5×10⁻⁴ sec⁻¹, 1×10⁻⁴ sec⁻¹, 5×10⁻⁵ sec⁻¹, 1×10⁻⁵ sec⁻¹, 5×10⁻⁶sec⁻¹, 1×10⁻⁶ sec⁻¹, 5×10⁻⁷ sec⁻¹, or 1×10⁻⁷ sec⁻¹.

In other embodiments, antibodies of the invention bind to B7-relatedpolypeptides of the invention or fragments or variants thereof with anon rate (Kon) of greater than or equal to 1×10³ M⁻¹sec⁻¹, 5×10³M⁻¹sec⁻¹, 1×10⁴ M⁻¹sec⁻¹, or 5×10⁴ M⁻¹sec⁻¹. More preferably, antibodiesof the invention bind to B7-related polypeptides of the invention orfragments or variants thereof with an on rate greater than or equal to1×10⁵ M⁻¹sec⁻¹, 5×10⁵ M⁻¹sec⁻¹, 1×10⁶ M⁻¹sec⁻¹, 5×10⁻⁶ M⁻¹sec⁻¹, or1×10⁻⁷ M⁻¹sec⁻¹.

The present invention also provides antibodies that competitivelyinhibit the binding of an antibody to an epitope of the invention asdetermined by any method known in the art for determining competitivebinding, for example, the immunoassays as described herein. In preferredembodiments, the antibody competitively inhibits binding to an epitopeby at least 95%, at least 90%, at least 85%, at least 80%, at least 75%,at least 70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonistsof the B7-related polypeptides of the present invention. For example,the present invention includes antibodies that disrupt theintra-cellular or inter-cellular activity, or interactions, of thepolypeptides of the invention either partially or fully. The inventionincludes BSL1, BSL2, and BSL3-specific antibodies and antibody specificfor the corresponding BSL-binding partner complexes. The invention alsoincludes BSL1-, BSL2-, or BSL3-specific antibodies which do not preventinteraction with a cognate binding partner (e.g., ligand), but doprevent activation. Activation (i.e., signaling) can be determined bytechniques described herein or as otherwise known in the art. Inspecific embodiments, antibodies are provided that inhibit BSL1, BSL2,or BSL3 binding activity or activation activity by at least 95%, atleast 90%, at least 85%, at least 80%, at least 75%, at least 70%, atleast 60%, or at least 50% of the activity in the absence of theantibody.

In another embodiment of the present invention, antibodies thatimmunospecifically bind to a B7-related polypeptide of the invention ora fragment or variant thereof, comprise a polypeptide having the aminoacid sequence of any one of the heavy chains expressed by an anti-BSL1,-BSL2, or -BSL3 antibody-expressing cell line of the invention, and/orany one of the light chains expressed by an anti-BSL1, -BSL2, or -BSL3antibody-expressing cell line of the invention. In another embodiment ofthe present invention, antibodies that immunospecifically bind to aB7-related polypeptide of the invention or a fragment or variantthereof, comprise a polypeptide having the amino acid sequence of anyone of the V_(H) domains of a heavy chain expressed by an anti-BSL1,-BSL2, or -BSL3 antibody-expressing cell line, and/or any one of theV_(L) domains of a light chain expressed by an anti-BSL1, -BSL2, or-BSL3 antibody-expressing cell line. In preferred embodiments,antibodies of the present invention comprise the amino acid sequence ofa V_(H) domain and V_(L) domain expressed by a single anti-BSL1, -BSL2,or -BSL3 antibody-expressing cell line. In alternative embodiments,antibodies of the present invention comprise the amino acid sequence ofa V_(H) domain and a V_(L) domain expressed by two different anti-BSL1,-BSL2, or -BSL3 antibody-expressing cell lines.

Molecules comprising, or alternatively consisting of, antibody fragmentsor variants of the V_(H) and/or V_(L) domains expressed by an anti-BSL1,-BSL2, or -BSL3 antibody-expressing cell line that immunospecificallybind to a B7-related polypeptide of the invention are also encompassedby the invention, as are nucleic acid molecules encoding these V_(H) andV_(L) domains, molecules, fragments and/or variants.

The present invention also provides antibodies that immunospecificiallybind to a polypeptide, or polypeptide fragment or variant of aB7-related polypeptide such as BSL1 (e.g., SEQ ID NO:2), BSL2 (e.g., SEQID NO:7, 11, or 13), or BSL3 (e.g., SEQ ID NO:15), wherein saidantibodies comprise, or alternatively consist of, a polypeptide havingan amino acid sequence of any one, two, three, or more of the V_(H) CDRscontained in a heavy chain expressed by one or more anti-BSL1, -BSL2, or-BSL3 antibody expressing cell lines. In particular, the inventionprovides antibodies that immunospecifically bind to a B7-relatedpolypeptide of the invention, comprising, or alternatively consistingof, a polypeptide having the amino acid sequence of a V_(H) CDR1contained in a heavy chain expressed by one or more anti-BSL1, -BSL2, or-BSL3 antibody expressing cell lines. In another embodiment, antibodiesthat immunospecifically bind to a B7-=related polypeptide of theinvention, comprise, or alternatively consist of, a polypeptide havingthe amino acid sequence of a V_(H) CDR2 contained in a heavy chainexpressed by one or more anti-BSL1, -BSL2, or -BSL3 antibody expressingcell lines. In a preferred embodiment, antibodies thatimmunospecifically bind to a B7-related polypeptide of the invention,comprise, or alternatively consist of, a polypeptide having the aminoacid sequence of a V_(H) CDR3 contained in a heavy chain expressed byone or more anti-BSL1, -BSL2, or -BSL3 antibody expressing cell line ofthe invention. Molecules comprising, or alternatively consisting of,these antibodies, or antibody fragments or variants thereof, thatimmunospecifically bind to a B7-related polypeptide (e.g., BSL1, BSL2,or BSL3) or a polypeptide fragment or variant thereof are alsoencompassed by the invention, as are nucleic acid molecules encodingthese antibodies, molecules, fragments and/or variants.

The present invention also provides antibodies that immunospecificiallybind to a polypeptide, or polypeptide fragment or variant of aB7-related polypeptide disclosed herein, wherein said antibodiescomprise, or alternatively consist of, a polypeptide having an aminoacid sequence of any one, two, three, or more of the V_(L) CDRscontained in a heavy chain expressed by one or more anti-BSL1, -BSL2, or-BSL3 antibody expressing cell lines of the invention. In particular,the invention provides antibodies that immunospecifically bind to aB7-related polypeptide disclosed herein, comprising, or alternativelyconsisting of, a polypeptide having the amino acid sequence of a V_(L)CDR1 contained in a heavy chain expressed by one or more anti-BSL1,-BSL2, or -BSL3 antibody-expressing cell lines of the invention. Inanother embodiment, antibodies that immunospecifically bind to aB7-related polypeptide of the invention, comprise, or alternativelyconsist of, a polypeptide having the amino acid sequence of a V_(L) CDR2contained in a heavy chain expressed by one or more anti-BSL1, -BSL2, or-BSL3 antibody-expressing cell lines of the invention. In a preferredembodiment, antibodies that immunospecifically bind to a B7-relatedpolypeptide of the invention, comprise, or alternatively consist of apolypeptide having the amino acid sequence of a V_(L) CDR3 contained ina heavy chain expressed by one or more BSL1, -BSL2, or -BSL3antibody-expressing cell lines of the invention. Molecules comprising,or alternatively consisting of, these antibodies, or antibody fragmentsor variants thereof, that immunospecifically bind to a B7-relatedpolypeptide such as BSL1 (e.g., SEQ ID NO:2), BSL2 (e.g., SEQ ID NO:7,11, or 13), or BSL3 (e.g., SEQ ID NO:15), or a polypeptide fragment orvariant thereof are also encompassed by the invention, as are nucleicacid molecules encoding these antibodies, molecules, fragments and/orvariants.

The present invention also provides antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants) that immunospecifically bind to a B7-related polypeptide, orpolypeptide fragment or variant disclosed herein, wherein the antibodiescomprise, or alternatively consist of, one, two, three, or more V_(H)CDRs, and one, two, three or more V_(L) CDRs, as contained in a heavychain or light chain expressed by one or more anti-BSL1, -BSL2, or -BSL3antibody-expressing cell lines of the invention. In particular, theinvention provides antibodies that immunospecifically bind to apolypeptide or polypeptide fragment or variant of a B7-relatedpolypeptide disclosed herein, wherein the antibodies comprise, oralternatively consist of, a V_(H) CDR1 and a V_(L) CDR1, a V_(H) CDR1and a V_(L) CDR2, a V_(H) CDR1 and a V_(L) CDR3, a V_(H) CDR2 and aV_(L) CDR1, V_(H) CDR2 and V_(L) CDR2, a V_(H) CDR2 and a V_(L) CDR3, aV_(H) CDR3 and a V_(H) CDR1, a V_(H) CDR3 and a V_(L) CDR2, a V_(H) CDR3and a V_(L) CDR3, or any combination thereof, of the V_(H) CDRs andV_(L) CDRs contained in a heavy chain or light chain immunoglobulinmolecule expressed by one or more anti-BSL1, -BSL2, or -BSL3antibody-expressing cell lines of the invention. In a preferredembodiment, one or more of these combinations are from a singleanti-BSL1, -BSL2, or -BSL3 antibody-expressing cell line of theinvention. Molecules comprising, or alternatively consisting of,fragments or variants of these antibodies that immunospecifically bindto a B7-related polypeptide such as BSL1 (e.g., SEQ ID NO:2), BSL2(e.g., SEQ ID NO:7, 11, or 13), or BSL3 (e.g., SEQ ID NO:15) are alsoencompassed by the invention, as are nucleic acid molecules encodingthese antibodies, molecules, fragments or variants.

The present invention also provides nucleic acid molecules, generallyisolated, encoding an antibody of the invention (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof). In a specific embodiment, a nucleic acid molecule ofthe invention encodes an antibody (including molecules comprising, oralternatively consisting of, antibody fragments or variants thereof),comprising, or alternatively consisting of, a V_(H) domain having anamino acid sequence of any one of the V_(H) domains of a heavy chainexpressed by an anti-BSL1, -BSL2, or -BSL3 antibody-expressing cell lineof the invention and a V_(L) domain having an amino acid sequence of alight chain expressed by an anti-BSL1, -BSL2, or -BSL3antibody-expressing cell line of the invention. In another embodiment, anucleic acid molecule of the invention encodes an antibody (includingmolecules comprising, or alternatively consisting of, antibody fragmentsor variants thereof), comprising, or alternatively consisting of, aV_(H) domain having an amino acid sequence of any one of the V_(H)domains of a heavy chain expressed by an anti-BSL1, -BSL2, or -BSL3antibody-expressing cell line of the invention, or a V_(L) domain havingan amino acid sequence of a light chain expressed by an anti-BSL1,-BSL2, or -BSL3 antibody-expressing cell line of the invention.

The present invention also provides antibodies that comprise, oralternatively consist of, variants (including derivatives) of theantibody molecules (e.g., the V_(H) domains and/or V_(L) domains)described herein, which antibodies immunospecifically bind to aB7-related polypeptide or fragment or variant thereof, as disclosedherein.

Standard techniques known to those of skill in the art can be used tointroduce mutations in the nucleotide sequence encoding a molecule ofthe invention, including, for example, site-directed mutagenesis andPCR-mediated mutagenesis which result in amino acid substitutions.Preferably the molecules are immunoglobulin molecules. Also, preferably,the variants (including derivatives) encode less than 50 amino acidsubstitutions, less than 40 amino acid substitutions, less than 30 aminoacid substitutions, less than 25 amino acid substitutions, less than 20amino acid substitutions, less than 15 amino acid substitutions, lessthan 10 amino acid substitutions, less than 5 amino acid substitutions,less than 4 amino acid substitutions, less than 3 amino acidsubstitutions, or less than 2 amino acid substitutions, relative to thereference V_(H) domain, V_(H) CDR1, V_(H) CDR2, V_(H) CDR3, V_(L)domain, V_(L) CDR1, V_(L) CDR2, or V_(L) CDR3.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a side chain witha similar charge. Families of amino acid residues having side chainswith similar charges have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis. The resultant mutants can be screened forbiological activity to identify mutants that retain activity.

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody molecule. Introducedmutations can be silent or neutral missense mutations, i.e., have no, orlittle, effect on an antibody's ability to bind antigen. These types ofmutations can be useful to optimize codon usage, or to improve hybridomaantibody production. Alternatively, non-neutral missense mutations canalter an antibody's ability to bind antigen. The location of most silentand neutral missense mutations is likely to be in the framework regions,while the location of most non-neutral missense mutations is likely tobe in the CDRs, although this is not an absolute requirement. One ofskill in the art is able to design and test mutant molecules withdesired properties, such as no alteration in antigen binding activity oralteration in binding activity (e.g., improvements in antigen bindingactivity or change in antibody specificity). Following mutagenesis, theencoded protein may routinely be expressed and the functional and/orbiological activity of the encoded protein can be determined usingtechniques described herein or by routinely modifying techniques knownand practiced in the art.

In a specific embodiment, an antibody of the invention (including amolecule comprising, or alternatively consisting of, an antibodyfragment or variant thereof), that immunospecifically binds toB7-related polypeptides or fragments or variants disclosed herein,comprises, or alternatively consists of, an amino acid sequence encodedby a nucleotide sequence that hybridizes to a nucleotide sequence thatis complementary to that encoding one of the V_(H) or V_(L) domainsexpressed by one or more anti-BSL1, -BSL2, or -BSL3 antibody-expressingcell lines of the invention, preferably under stringent conditions,e.g., hybridization to filter-bound DNA in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C. followed by one or more washes in0.2×SSC/0.1% SDS at about 50-65° C., preferably under highly stringentconditions, e.g., hybridization to filter-bound nucleic acid in 6×SSC atabout 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about68° C., or under other stringent hybridization conditions which areknown to those of skill in the art (see, for example, F. M. Ausubel etal., eds. (1989) Current Protocols in Molecular Biology, Vol. I, GreenPublishing Associates, Inc. and John Wiley & Sons, Inc., New York atpages 6.3.1-6.3.6 and 2.10.3). Nucleic acid molecules encoding theseantibodies are also encompassed by the invention.

It is well known within the art that polypeptides, or fragments orvariants thereof, with similar amino acid sequences often have similarstructure and many of the same biological activities. Thus, in oneembodiment, an antibody (including a molecule comprising, oralternatively consisting of, an antibody fragment or variant thereof),that immunospecifically binds to a B7-related polypeptide or fragmentsor variants of a B7-related polypeptide disclosed herein, comprises, oralternatively consists of, a V_(H) domain having an amino acid sequencethat is at least 35%, at least 40%, at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or at least 99% identicalto the amino acid sequence of a V_(H) domain of a heavy chain expressedby an anti-BSL1, -BSL2, or -BSL3 antibody-expressing cell line of theinvention.

In another embodiment, an antibody (including a molecule comprising, oralternatively consisting of, an antibody fragment or variant thereof),that immunospecifically binds to a B7-related polypeptide or fragmentsor variants of a B7-related polypeptide disclosed herein, comprises, oralternatively consists of, a V_(L) domain having an amino acid sequencethat is at least 35%, at least 40%, at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or at least 99% identicalto the amino acid sequence of a V_(L) domain of a light chain expressedby an anti-BSL1, -BSL2, or -BSL3 antibody-expressing cell line of theinvention.

The present invention also provides antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof), that down-regulate the cell-surface expression of aB7-related polypeptide of the invention, as determined by any methodknown in the art such as, for example, FACS analysis orimmunofluorescence assays. By way of a non-limiting hypothesis, suchdown-regulation may be the result of antibody induced internalization ofB7-related polypeptide of the invention. Such antibodies can comprise,or alternatively consist of, a portion (e.g., V_(H) CDR1, V_(H) CDR2,V_(H) CDR3, V_(L) CDR1, V_(L) CDR2, or V_(L) CDR3) of a V_(H) or V_(L)domain having an amino acid sequence of an antibody of the invention, ora fragment or variant thereof.

In another embodiment, an antibody that down-regulates the cell-surfaceexpression of a B7-related polypeptide of the invention comprises, oralternatively consists of, a polypeptide having the amino acid sequenceof a V_(H) domain of an antibody of the invention, or a fragment orvariant thereof and a V_(L) domain of an antibody of the invention, or afragment or variant thereof. In another embodiment, an antibody thatdown-regulates the cell-surface expression of a B7-related polypeptideof the invention comprises, or alternatively consists of, a polypeptidehaving the amino acid sequence of a V_(H) domain and a V_(L) domain froma single antibody (or scFv or Fab fragment) of the invention, orfragments or variants thereof. In another embodiment, an antibody thatdown-regulates the cell-surface expression of a B7-related polypeptideof the invention comprises, or alternatively consists of, a polypeptidehaving the amino acid sequence of a V_(H) domain of an antibody of theinvention, or a fragment or variant thereof. In another embodiment, anantibody that down-regulates the cell-surface expression of a B7-relatedpolypeptide of the invention comprises, or alternatively consists of, apolypeptide having the amino acid sequence of a V_(L) domain of anantibody of the invention, or a fragment or variant thereof.

In a preferred embodiment, an antibody that down-regulates thecell-surface expression of a B7-related polypeptide of the inventioncomprises, or alternatively consists of, a polypeptide having the aminoacid sequence of a V_(H) CDR3 of an antibody of the invention, or afragment or variant thereof. In another preferred embodiment, anantibody that down-regulates the cell-surface expression of a B7-relatedpolypeptide of the invention comprises, or alternatively consists of, apolypeptide having the amino acid sequence of a V_(L) CDR3 of anantibody of the invention, or a fragment or variant thereof. Nucleicacid molecules encoding these antibodies are also encompassed by theinvention.

In another preferred embodiment, an antibody that enhances the activityof a B7-related polypeptide, or a fragment or variant disclosed herein,comprises, or alternatively consists of, a polypeptide having the aminoacid sequence of a V_(L) CDR3 of an antibody of the invention, or afragment or variant thereof. Nucleic acid molecules encoding theseantibodies are also encompassed by the invention.

As nonlimiting examples, antibodies of the present invention can be usedto purify, detect, and target the polypeptides of the present invention,including both in vitro and in vivo diagnostic, detection, screening,and/or therapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe B7-related polypeptides of the present invention in biologicalsamples. (See, e.g., Harlow et al. (1988) Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, 2nd Ed., which isincorporated by reference herein in its entirety).

By way of another nonlimiting example, antibodies of the invention canbe administered to individuals as a form of passive immunization.Alternatively, antibodies of the present invention can be used forepitope mapping to identify the epitope(s) that are bound by theantibody. Epitopes identified in this way can, in turn, for example, beused as vaccine candidates, i.e., to immunize an individual to elicitantibodies against the naturally-occurring forms of one or moreB7-related polypeptides of the invention.

As discussed in more detail below, the antibodies of the presentinvention can be used either alone or in combination with othercompositions. The antibodies can further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus, or chemicallyconjugated (including covalent and non-covalent conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention can be recombinantly fused or conjugated to moleculesthat are useful as labels in detection assays and to effector moleculessuch as heterologous polypeptides, drugs, radionucleotides, or toxins.See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S.Pat. No. 5,314,995 and EP 396,387.

The antibodies of the invention include derivatives that are modified,i.e., by the covalent attachment of any type of molecule to theantibody. For example, without limitation, the antibody derivativesinclude antibodies that have been modified, e.g., by glycosylation,acetylation, pegylation, phosphorylation, amidation, derivatization byknown protecting/blocking groups, proteolytic cleavage, linkage to acellular ligand or other protein, etc. Any of numerous chemicalmodifications may be carried out by known techniques, including, but notlimited to, specific chemical cleavage, acetylation, formylation,metabolic synthesis of tunicamycin, etc. Additionally, the derivativecan contain one or more non-classical amino acids.

The antibodies of the present invention may be generated by any suitablemethod known in the art. Polyclonal antibodies directed against anantigen or immunogen of interest can be produced by various procedureswell known in the art. For example, a B7-related polypeptide or peptideof the invention can be administered to various host animals aselucidated above to induce the production of sera containing polyclonalantibodies specific for the antigen. Various adjuvants may be used toincrease the immunological response, depending on the host species;adjuvants include, but are not limited to, Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as BCG (bacille Calmette-Guerin)and corynebacterium parvum. Such adjuvants are also well known in theart.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art, including the use of hybridoma, recombinant and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques as known andpracticed in the art (as taught, for example, in Harlow et al. (1988)Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press,2nd Ed.; and Hammerling, et al.; (1981) Monoclonal Antibodies and T-CellHybridomas, Elsevier, N.Y., pages 563-681, the contents of which areincorporated herein by reference in their entireties). The term“monoclonal antibody” as used herein is not limited to antibodiesproduced through hybridoma technology. The term “monoclonal antibody”refers to an antibody that is derived from a single clone, including anyeukaryotic, prokaryotic, or phage clone, and not the method by which itis produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In anonlimiting example, mice can be immunized with a polypeptide or peptideof the invention, or with a cell expressing the polypeptide or peptide.Once an immune response is detected, e.g., antibodies specific for theantigen are detected in the sera of immunized mice, the spleen isharvested and splenocytes are isolated. The splenocytes are then fusedby well known techniques to any suitable myeloma cells, for examplecells from cell line SP2/0 or P3X63-AG8.653 available from the ATCC.Hybridomas are selected and cloned by limiting dilution techniques. Thehybridoma clones are then assayed by methods known in the art todetermine and select those cells that secrete antibodies capable ofbinding to a polypeptide of the invention. Ascites fluid, whichgenerally contains high levels of antibodies, can be generated byimmunizing mice with positive hybridoma clones.

Accordingly, the present invention encompasses methods of generatingmonoclonal antibodies, as well as the antibodies produced by thesemethods, comprising culturing a hybridoma cell secreting an antibody ofthe invention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with a B7-relatedpolypeptide or peptide antigen of the invention with myeloma cells andthen screening the hybridomas resulting from the fusion for hybridomaclones that secrete an antibody that binds to a polypeptide of theinvention such as BSL1 (e.g., SEQ ID NO:2), BSL2 (e.g., SEQ ID NO:7, 11,or 13), or BSL3 (e.g., SEQ I NO:15).

Another well known method for producing both polyclonal and monoclonalhuman B cell lines is transformation using Epstein Barr Virus (EBV).Protocols for generating EBV-transformed B cell lines are commonly knownin the art (see, for example, the protocol outlined in Chapter 7.22 ofColigan et al., Eds., (1994) Current Protocols in Immunology, John Wiley& Sons, NY, which is hereby incorporated by reference herein in itsentirety). The source of B cells for transformation is commonly humanperipheral blood, but B cells for transformation can also be obtainedfrom other sources including, but not limited to, lymph node, tonsil,spleen, tumor tissue, and infected tissues. Tissues are generallyprepared as single cell suspensions prior to EBV transformation. Inaddition, T-cells that may be present in the B cell samples can beeither physically removed or inactivated (e.g., by treatment withcyclosporin A). The removal of T-cells is often advantageous, becauseT-cells from individuals seropositive for anti-EBV antibodies cansuppress B cell immortalization by EBV. In general, a sample containinghuman B cells is inoculated with EBV and cultured for 3-4 weeks. Atypical source of EBV is the culture supernatant of the B95-8 cell line(ATCC; VR-1492). Physical signs of EBV transformation can generally beseen toward the end of the 3-4 week culture period.

By phase-contrast microscopy, transformed cells appear large, clear and“hairy”; they tend to aggregate in tight clusters of cells. Initially,EBV lines are generally polyclonal. However, over prolonged periods ofcell culture, EBV lines can become monoclonal as a result of theselective outgrowth of particular B cell clones. Alternatively,polyclonal EBV transformed lines can be subcloned (e.g., by limitingdilution) or fused with a suitable fusion partner and plated at limitingdilution to obtain monoclonal B cell lines. Suitable fusion partners forEBV transformed cell lines include mouse myeloma cell lines (e.g.,SP2/0, X63-Ag8.653), heteromyeloma cell lines (human×mouse; e.g.,SPAM-8, SBC-H20, and CB-F7), and human cell lines (e.g., GM 1500,SKO-007, RPMI 8226, and KR-4). Thus, the present invention also includesa method of generating polyclonal or monoclonal human antibodies againstpolypeptides of the invention or fragments thereof, comprisingEBV-transformation of human B cells.

Antibody fragments that recognize specific epitopes can be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F (ab′) 2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

Antibodies encompassed by the present invention can also be generatedusing various phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles that carry the polynucleotide sequences encoding them.In a particular embodiment, such phage can be utilized to displayantigen binding domains expressed from a repertoire or combinatorialantibody library (e.g., human or murine). Phage expressing an antigenbinding domain that binds to the antigen of interest can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured onto a solid surface or bead. Phage used in these methods aretypically filamentous phage including fd and M13 binding domainsexpressed from phage with Fab, Fv or disulfide stabilized Fv antibodydomains recombinantly fused to either the phage gene III or gene VIIIprotein. Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al. (1995) J. Immunol. Methods, 182:41-50; Ames et al. (1995) J.Immunol. Methods, 184:177-186; Kettleborough et al. (1994) Eur. J.Immunol. 24:952-958; Persic et al. (1997) Gene, 187:9-18; Burton et al.(1994) Advances in Immunology, 57:191-280; PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108, each of which is incorporated herein byreference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al. (1992) BioTechniques,12(6):864-869; Sawai et al. (1995) AJRI, 34:2634; and Better et al.(1988) Science, 240:1041-1043, which are hereby incorporated byreference herein in their entireties.

Examples of techniques that can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al. (1991) Methods Enzymol., 203:46-88; Shu et al.(1993) Proc. Natl. Acad. Sci. USA, 90:7995-7999; and Skerra et al.(1988) Science, 240:1038-1040. For some uses, including the in vivo useof antibodies in humans and in in vitro detection assays, it may bepreferable to use chimeric, humanized, or human antibodies. A chimericantibody is a molecule in which different portions of the antibody arederived from different animal species, such as antibodies having avariable region derived from a murine monoclonal antibody and a humanimmunoglobulin constant region. Methods for producing chimericantibodies are known in the art. (See, e.g., Morrison (1985) Science,229:1202; Oi et al. (1986) BioTechniques, 4:214; Gillies et al. (1989)J. Immunol. Methods, 125:191-202; and U.S. Pat. Nos. 5,807,715;4,816,567; and 4,816,397, which are incorporated herein by reference intheir entirety).

Humanized antibodies are antibody molecules from nonhuman speciesantibody that bind to the desired antigen and have one or morecomplementarity determining regions (CDRs) from the nonhuman species andframework regions from a human immunoglobulin molecule. Often, frameworkresidues in the human framework regions are substituted with thecorresponding residues from the CDR donor antibody to alter, preferablyimprove, antigen binding. These framework substitutions are identifiedby methods well known in the art, e.g., by modeling of the interactionsof the CDR and framework residues to identify framework residuesimportant for antigen binding, and by sequence comparison to identifyunusual framework residues at particular positions. (See, e.g., Queen etal., U.S. Pat. No. 5,585,089; and Riechmann et al. (1988) Nature,332:323, which are incorporated herein by reference in theirentireties). Antibodies can be humanized using a variety of techniquesknown in the art, including, for example, CDR-grafting (EP 239,400; PCTpublication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and5,585,089); veneering or resurfacing (EP 592,106; EP 519,596; Padlan(1991) Molecular Immunology, 28:489-498; Studnicka et al. (1994) ProteinEngineering, 7(6):805-814; Roguska et al. (1994) Proc. Natl. Acad. Sci.USA, 91:969-973; and chain shuffling (U.S. Pat. No. 5,565,332).

Completely human antibodies can be made by a variety of methods known inthe art, including the phage display methods described above, usingantibody libraries derived from human immunoglobulin sequences. Seealso, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO96/33735, and WO 91/10741; each of which is incorporated herein byreference in its entirety.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients, so as to avoid or alleviate immune reactionto foreign protein. Human antibodies can be made by a variety of methodsknown in the art, including the phage display methods described above,using antibody libraries derived from human immunoglobulin sequences.See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publicationsWO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO96/33735, and WO 91/10741; each of which is incorporated herein byreference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes can be introduced randomly, orby homologous recombination, into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells, in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes can be rendered nonfunctional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of theJ_(H) region prevents endogenous antibody production. The modifiedembryonic stem cells are expanded and microinjected into blastocysts toproduce chimeric mice. The chimeric mice are then bred to producehomozygous offspring that express human antibodies. The transgenic miceare immunized in the normal fashion with a selected antigen, e.g., allor a portion of a polypeptide of the invention.

Monoclonal antibodies directed against the antigen can be obtained fromthe immunized transgenic mice using conventional hybridoma technology.The human immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation.

Thus, using such a technique, it is possible to produce useful humanIgG, IgA, IgM and IgE antibodies. For an overview of the technology forproducing human antibodies, see Lonberg and Huszar (1995) Intl. Rev.Immunol. 13:65-93. For a detailed discussion of the technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies, see, e.g., PCT publications WO 98/24893;WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877;U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016;5,545,806; 5,814,318; 5,885,793; 5,916,771; 5,939,598; 6,075,181; and6,114,598, which are incorporated by reference herein in their entirety.In addition, companies such as Abgenix, Inc. (Fremont, Calif.) andGenpharm (San Jose, Calif.) can be engaged to provide human antibodiesdirected against a selected antigen using technology similar to theabove described technologies.

Completely human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection”. In thisapproach, a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope (Jespers et al. (1988) BioTechnology,12:899-903).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotypic antibodies that “mimic”B7-related polypeptides of the invention using techniques well known tothose skilled in the art. (See, e.g., Greenspan and Bona (1989) FASEB J.7(5):437-444 and Nissinoff (1991) J. Immunol. 147(8):2429-2438). Forexample, antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” one or moreof the BSL-1, BSL2, or BSL2 polypeptide domains and, as a consequence,bind to and neutralize the polypeptide and/or its binding partner, e.g.,in therapeutic regimens. Such neutralizing anti-idiotypes or Fabfragments of such anti-idiotypes can be used to neutralize polypeptideactivity. For example, such anti-idiotypic antibodies can be used tobind a polypeptide of the invention and/or to bind its, and therebyactivate or block its biological activity.

Intrabodies are antibodies, often scFvs, that are expressed from arecombinant nucleic acid molecule and are engineered to be retainedintracellularly (e.g., retained in the cytoplasm, endoplasmic reticulum,or periplasm of the host cells). Intrabodies can be used, for example,to ablate the function of a protein to which the intrabody binds. Theexpression of intrabodies can also be regulated through the use ofinducible promoters in the nucleic acid expression vector comprisingnucleic acid encoding the intrabody. Intrabodies of the invention can beproduced using methods known in the art, such as those disclosed andreviewed in Chen et al. (1994) Hum. Gene Ther. 5:595-601; W. A. Marasco(1997) Gene Ther. 4:11-15; Rondon and Marasco (1997) Annu. Rev.Microbiol. 51:257-283; Proba et al. (1998) J. Mol. Biol. 275:245-253;Cohen et al. (1998) Oncogene, 17:2445-2456; Ohage and Steipe (1999) J.Mol. Biol. 291:1119-1128; Ohage et al. (1999) J. Mol. Biol.291:1129-1134; Wirtz and Steipe (1999) Protein Sci. 8:2245-2250; Zhu etal. (1999) J. Immunol. Methods, 231:207-222.

XenoMouse Technology Antibodies in accordance with the invention arepreferably pre0pared by the utilization of a transgenic mouse that has asubstantial portion of the human antibody producing genome inserted, butthat is rendered deficient in the production of endogenous murineantibodies (e.g., XenoMouse strains available from Abgenix Inc.,Fremont, Calif.). Such mice are capable of producing humanimmunoglobulin molecules and antibodies and are virtually deficient inthe production of murine immunoglobulin molecules and antibodies.Technologies utilized for achieving the same are disclosed in thepatents, applications, and references disclosed herein.

The ability to clone and reconstruct megabase-sized human loci in YACsand to introduce them into the mouse germline provides a powerfulapproach to elucidating the functional components of very large orcrudely mapped loci, as well as generating useful models of humandisease. Furthermore, the utilization of such technology forsubstitution of mouse loci with their human equivalents could provideunique insights into the expression and regulation of human geneproducts during development, their communication with other systems, andtheir involvement in disease induction and progression. An importantpractical application of such a strategy is the “humanization” of themouse humoral immune system. Introduction of human immunoglobulin (Ig)loci into mice in which the endogenous Ig genes have been inactivatedoffers the opportunity to study the mechanisms underlying programmedexpression and assembly of antibodies as well as their role in B celldevelopment. Furthermore, such a strategy could provide an ideal sourcefor the production of fully human monoclonal antibodies: an importantmilestone toward fulfilling the promise of antibody therapy in humandisease.

Fully human antibodies are expected to minimize the immunogenic andallergic responses intrinsic to mouse or mouse-derivatized monoclonalantibodies and thus to increase the efficacy and safety of theadministered antibodies. The use of fully human antibodies can beexpected to provide a substantial advantage in the treatment of chronicand recurring human diseases, such as cancer, which require repeatedantibody administrations.

One approach toward this goal was to engineer mouse strains deficient inmouse antibody production to harbor large fragments of the human Ig lociin anticipation that such mice would produce a large repertoire of humanantibodies in the absence of mouse antibodies. Large human Ig fragmentswould preserve the large variable gene diversity as well as the properregulation of antibody production and expression. By exploiting themouse machinery for antibody diversification and selection and the lackof immunological tolerance to human proteins, the reproduced humanantibody repertoire in these mouse strains should yield high affinityantibodies against any antigen of interest, including human antigens.Using the hybridoma technology, antigen-specific human monoclonalantibodies with the desired specificity could be readily produced andselected.

This general strategy was demonstrated in connection with the generationof the first “XenoMouseT” strains as published in 1994. See Green et al.(1994) Nature Genetics, 7:13-21. The XenoMouse strains were engineeredwith yeast artificial chromosomes (YACS) containing 245-kb and10,190-kb-sized germline configuration fragments of the human heavychain locus and kappa light chain locus, respectively, which containedcore variable and constant region sequences. Id. The human Ig containingYACs proved to be compatible with the mouse system for bothrearrangement and expression of antibodies and were capable ofsubstituting for the inactivated mouse Ig genes. This was demonstratedby their ability to induce B-cell development, to produce an adult-likehuman repertoire of fully human antibodies, and to generateantigen-specific human monoclonal antibodies. These results alsosuggested that introduction of larger portions of the human Ig locicontaining greater numbers of V genes, additional regulatory elements,and human Ig constant regions might recapitulate substantially the fullrepertoire that is characteristic of the human humoral response toinfection and immunization. The work of Green et al. was recentlyextended to the introduction of greater than approximately 80% of thehuman antibody repertoire through the use of megabase-sized, germlineconfiguration YAC fragments of the human heavy chain loci and kappalight chain loci, respectively, to produce XenoMouse mice. See Mendez etal. (1997) Nature Genetics, 15:146-156; Green and Jakobovits (1998) J.Exp. Med. 188:483-495; and Green (1999) J. Immunol. Methods, 231:11-23,the disclosures of which are hereby incorporated herein by reference.

Human anti-mouse antibody (HAMA) responses have led the industry toprepare chimeric or otherwise humanized antibodies. While chimericantibodies typically are comprised of a human constant region and amurine variable region, it is expected that certain human anti-chimericantibody (HACA) responses will be observed, particularly in treatmentsinvolving chronic or multi-dose utilizations of the antibody. Thus, itis desirable to provide fully human antibodies against B7-relatedpolypeptides of the invention in order to vitiate concerns and/oreffects of HAMA or HACA responses.

Polypeptide antibodies of the invention may be chemically synthesized orproduced through the use of recombinant expression systems. Accordingly,the invention further embraces polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedsupra, to polynucleotides that encode an antibody, preferably, anantibody that specifically binds to a polypeptide of the invention,preferably, an antibody that binds to a polypeptide having the aminoacid sequence of one or more of the B7-related sequences as set forth inFIGS. 1-6.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody can be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al. (1994)BioTechniques, 17:242), which, briefly, involves the synthesis ofoverlapping oligonucleotides containing portions of the sequenceencoding the antibody, the annealing and ligating of thoseoligonucleotides, and then the amplification of the ligatedoligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody can be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin can be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, (or a nucleic acid, preferably poly(A)⁺ RNA, isolated from), anytissue or cells expressing the antibody, such as hybridoma cellsselected to express an antibody of the invention by PCR amplificationusing synthetic primers hybridizable to the 3′ and 5′ ends of thesequence. Alternatively, cloning using an oligonucleotide probe specificfor the particular gene sequence to identify, e.g., a cDNA clone from acDNA library that encodes the antibody can be employed. Amplifiednucleic acids generated by PCR can then be cloned into replicablecloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody are determined, the nucleotide sequence of the antibody canbe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al. (1990) Molecular Cloning, A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; and Ausubel etal., eds., (1998) Current Protocols in Molecular Biology, John Wiley &Sons, NY, which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example, to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains can be inspected to identify the sequencesof the CDRs by methods that are well known in the art, e.g., bycomparison to known amino acid sequences of other heavy and light chainvariable regions, to determine the regions of sequence hypervariability.Using routine recombinant DNA techniques, one or more of the CDRs can beinserted within framework regions, e.g., into human framework regions,to humanize a non-human antibody, as described supra. The frameworkregions can be naturally occurring or consensus framework regions, andpreferably, are human framework regions (see, e.g., Chothia et al.(1998) J. Mol. Biol. 278:457-479 for a listing of human frameworkregions).

Preferably, the polynucleotide generated by the combination of theframework regions and CDRs encodes an antibody that specifically bindsto a B7-related polypeptide of the invention. Also preferably, asdiscussed supra, one or more amino acid substitutions can be made withinthe framework regions; such amino acid substitutions are performed withthe goal of improving binding of the antibody to its antigen. Inaddition, such methods can be used to make amino acid substitutions ordeletions of one or more variable region cysteine residues participatingin an intrachain disulfide bond to generate antibody molecules lackingone or more intrachain disulfide bonds. Other alterations to thepolynucleotide are encompassed by the present invention and are withinthe skill of the art.

For some uses, such as for in vitro affinity maturation of an antibodyof the invention, it is useful to express the V_(H) and V_(L) domains ofthe heavy and light chains of one or more antibodies of the invention assingle chain antibodies, or Fab fragments, in a phage display libraryusing phage display methods as described supra. For example, the cDNAsencoding the V_(H) and V_(L) domains of one or more antibodies of theinvention can be expressed in all possible combinations using a phagedisplay library, thereby allowing for the selection of V_(H)/V_(L)combinations that bind to the B7-related polypeptides according to thepresent invention with preferred binding characteristics such asimproved affinity or improved off rates. In addition, V_(H) and V_(L)segments, particularly, the CDR regions of the V_(H) and V_(L) domainsof one or more antibodies of the invention, can be mutated in vitro.Expression of V_(H) and V_(L) domains with “mutant” CDRs in a phagedisplay library allows for the selection of V_(H)/V_(L) combinationsthat bind to B7-related polypeptides of the invention with preferredbinding characteristics such as improved affinity or improved off rates.

In phage display methods, functional antibody domains are displayed onthe surface of phage particles that carry the polynucleotide sequencesencoding them. In particular, DNA sequences encoding the V_(H) and V_(L)domains are amplified from animal cDNA libraries (e.g., human or murinecDNA libraries of lymphoid tissues) or from synthetic cDNA libraries.The DNA encoding the V_(H) and V_(L) domains are joined together by anscFv linker by PCR and cloned into a phagemid vector (e.g., p CANTAB 6or pComb 3 HSS). The vector is introduced into E. coli viaelectroporation and the E. coli is infected with helper phage. Phageused in these methods are typically filamentous phage, including fd andM13, and the V_(H) and V_(L) domains are usually recombinantly fusedeither to the phage gene III or gene VIII. Phage expressing an antigenbinding domain that binds to an antigen of interest (i.e., a B7-relatedpolypeptide of the invention or a fragment thereof) can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured onto a solid surface or bead.

The antibodies according to the invention can be produced by any methodknown in the art for the synthesis of antibodies, in particular, bychemical synthesis, by intracellular immunization (i.e., intrabodytechnology), or preferably, by recombinant expression techniques.Methods of producing antibodies include, but are not limited to,hybridoma technology, EBV transformation, and other methods discussedherein as well as through the use recombinant DNA technology, asdiscussed below.

Recombinant expression of an antibody of the invention, or fragment,derivative, variant or analog thereof, (e.g., a heavy or light chain ofan antibody of the invention or a single chain antibody of theinvention), requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule can be produced by recombinant DNAtechnology using techniques well known in the art. Methods for preparinga protein by expressing a polynucleotide encoding an antibody aredescribed herein. Methods that are well known to those skilled in theart can be used to construct expression vectors containing antibodycoding sequences and appropriate transcriptional and translationalcontrol signals. These methods include, for example, in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. The invention, thus embraces replicable vectorscomprising a nucleotide sequence encoding an antibody molecule of theinvention, or a heavy or light chain thereof, or a heavy or light chainvariable domain, operably linked to a promoter. Such vectors can includethe nucleotide sequence encoding the constant region of the antibodymolecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of theantibody can be cloned into such a vector for expression of the entireheavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, or a single chainantibody of the invention, operably linked to a heterologous promoter.In preferred embodiments for the expression of double-chainedantibodies, vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host expression vector systems can be utilized to expressthe antibody molecules of the invention. Such expression systemsrepresent vehicles by which the coding sequences of interest can beexpressed, their encoded products produced and subsequently purified.These systems also represent cells that can, when transformed ortransfected with the appropriate nucleotide coding sequences, express anantibody molecule of the invention in situ. Cell expression systemsinclude, but are not limited, to microorganisms such as bacteria (e.g.,E. coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces or Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic virus(TMV)), transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3, NSO cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as E. coli, and morepreferably, eukaryotic cells, especially for the expression of wholerecombinant antibody molecules, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary (CHO) cells, in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus, is an effective expression system for antibodies(Foecking et al. (1986) Gene, 45:101; Cockett et al. (1990)BioTechnology, 8:2).

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced for the generation of pharmaceuticalcompositions of an antibody molecule, for example, vectors that directthe expression of high levels of fusion protein products that arereadily purified are often desirable. Such vectors include, but are notlimited to, the E. coli expression vector pUR278 (Ruther et al. (1983)EMBO J. 2:1791), in which the antibody coding sequence can be ligatedindividually into the vector in-frame with the lacZ coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye (1985)Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster (1989) J. Biol.Chem. 24:5503-5509; and the like). pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera figuriperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral based expression systems canbe utilized. In cases in which an adenovirus is used as an expressionvector, the antibody coding sequence of interest can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) results in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts. (See, e.g., Loganand Shenk (1984) Proc. Natl. Acad. Sci. USA, 81:355-359). Specificinitiation signals can also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in-phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression canbe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al. (1987)Methods in Enzymol. 153:51-544).

In addition, a host cell strain can be chosen to modulate the expressionof the inserted sequences, or modify and process the gene product in thespecific fashion desired. Such modifications (e.g., glycosylation) andprocessing (e.g., cleavage) of protein products can be important for thefunction of the protein.

Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins and geneproducts. Appropriate cell lines or host systems can be chosen to ensurethe correct modification and processing of the foreign proteinexpressed. To this end, eukaryotic host cells that possess the cellularmachinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product can be used. Suchmammalian host cells include, but are not limited to, CHO, VERY, BHK,HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer celllines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, andnormal mammary gland cell lines such as, for example, CRL7030 andHs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express theantibody molecule can be engineered. Rather than using expressionvectors that contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoters, enhancer sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, such geneticallyengineered cells can be allowed to grow for 1-2 days in an enrichedmedium, and then are typically replated in a selective medium. Aselectable marker in the recombinant plasmid confers resistance to theselection and allows cells to stably integrate the plasmid into theirchromosomes and grow to form foci which, in turn, can be cloned andexpanded into cell lines. This method can advantageously be used toengineer cell lines expressing the antibody molecule. Such engineeredcell lines are particularly useful in screening and evaluation ofcompounds that interact directly or indirectly with the antibodymolecule.

A number of selection systems can be used, including but not limited to,herpes simplex virus thymidine kinase (HSV TK), (Wigler et al. (1977)Cell, 11:223), hypoxanthine-guanine phosphoribosyltransferase (HGPRT),(Szybalska and Szybalski (1992) Proc. Natl. Acad. Sci. USA, 48:202), andadenine phosphoribosyltransferase (Lowy et al. (1980) Cell, 22:817)genes can be employed in tk−, hgprt−, or aprt− cells (APRT),respectively.

In addition, anti-metabolite resistance can be used as the basis ofselection for the following genes: dhfr, which confers resistance tomethotrexate (Wigler et al. (1980) Proc. Natl. Acad. Sci. USA, 77:357;and O'Hare et al. (1981) Proc. Natl. Acad. Sci. USA, 78:1527); gpt,which confers resistance to mycophenolic acid (Mulligan and Berg (1981)Proc. Natl. Acad. Sci. USA, 78:2072); neo, which confers resistance tothe aminoglycoside G418 (Clinical Pharmacy, 12:488-505; Wu and Wu (1991)Biotherapy, 3:87-95; Tolstoshev (1993) Ann. Rev. Pharmacol. Toxicol.32:573-596; Mulligan (1993) Science, 260:926-932; Anderson (1993) Ann.Rev. Biochem. 62:191-21; May (1993) TIB TECH, 11(5):155-215; and hygro,which confers resistance to hygromycin (Santerre et al. (1984) Gene,30:147). Methods commonly known in the art of recombinant DNA technologycan be routinely applied to select the desired recombinant clone; suchmethods are described, for example, in Ausubel et al., eds., (1990)Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler(1990) Gene Transfer and Expression, A Laboratory Manual, StocktonPress, NY; in Chapters 12 and 13, Dracopoli et al., eds., (1994) CurrentProtocols in Human Genetics, John Wiley & Sons, NY; Colberre-Garapin etal. (1981) J. Mol. Biol. 150:1, which are incorporated by referenceherein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel (1987) The Useof Vectors Based on Gene Amplification for the Expression of ClonedGenes in Mammalian Cells In DNA Cloning, Vol. 3. Academic Press, NewYork). When a marker in the vector system expressing an antibody isamplifiable, an increase in the level of inhibitor present in the hostcell culture will increase the number of copies of the marker gene.Since the amplified region is associated with the antibody gene,production of the antibody will also increase (Crouse et al. (1983) Mol.Cell. Biol. 3:257).

Vectors that use glutamine synthase (GS) or DHFR as the selectablemarkers can be amplified in the presence of the drugs methioninesulphoximine or methotrexate, respectively. An advantage of glutaminesynthase based vectors is the availability of cell lines (e.g., themurine myeloma cell line, NSO) which are glutamine synthase negative.Glutamine synthase expression systems can also function in glutaminesynthase expressing cells (e.g. Chinese Hamster Ovary (CHO) cells) byproviding additional inhibitor to prevent the functioning of theendogenous gene.

Vectors that express glutamine synthase as the selectable markerinclude, but are not limited to, the pEE6 expression vector (describedin Stephens and Cockett (1989) Nucl. Acids. Res. 17:7110). A glutaminesynthase expression system and components thereof are detailed in PCTpublications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; andWO91/06657, which are incorporated by reference herein in theirentireties. In addition, glutamine synthase expression vectors that canbe used in accordance with the present invention are commerciallyavailable from suppliers, including, for example, Lonza Biologics, Inc.(Portsmouth, N.H.). The expression and production of monoclonalantibodies using a GS expression system in murine myeloma cells has beendescribed (Bebbington et al. (1992) BioTechnology, 10:169 and in Bibliaand Robinson (1995) Biotechnol. Prog. 11:1, which are incorporated byreference herein in their entireties).

A host cell can be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors can contain identical selectable markers, which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector can be used which encodes, and is capable of expressing,both the heavy and light chain polypeptides. In such situations, thelight chain should be placed before the heavy chain to avoid an excessof toxic free heavy chain (Proudfoot (1986) Nature, 322:52; Kohler(1980) Proc. Natl. Acad. Sci. USA, 77:2197). The coding sequences forthe heavy and light chains can comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized, or recombinantly expressed, it can bepurified by any method known in the art for the purification of animmunoglobulin or polypeptide molecule, for example, by chromatography(e.g., ion exchange, affinity, particularly by affinity for the specificantigen, Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

The present invention encompasses antibodies that are recombinantlyfused or chemically conjugated (including both covalently andnon-covalently conjugated) to a polypeptide (or portion thereof,preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 contiguousamino acids of the polypeptide) of the present invention, such as BSL1(e.g., SEQ ID NO:2), BSL2 (e.g., SEQ ID NO:7, 11, or 13), or BSL3 (e.g.,SEQ ID NO:15), to generate fusion proteins. The fusion does notnecessarily need to be direct, but can occur through linker sequences.The antibodies can be specific for antigens other than polypeptides (orportions thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90or 100 contiguous amino acids of the polypeptide of the presentinvention. For example, antibodies can be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors.

Polypeptides and/or antibodies of the present invention (includingfragments or variants thereof) can be fused to either the N-terminal orC-terminal end of the heterologous protein (e.g., immunoglobulin Fcpolypeptide or human serum albumin polypeptide). Antibodies of theinvention can also be fused to albumin (including, but not limited to,recombinant human serum albumin (see, e.g., U.S. Pat. No. 5,876,969,issued Mar. 2, 1999; EP Patent 0 413 622; and U.S. Pat. No. 5,766,883,issued Jun. 16, 1998, incorporated herein by reference in theirentirety), resulting in chimeric polypeptides. In a preferredembodiment, polypeptides and/or antibodies of the present invention(including fragments or variants thereof) are fused with the mature formof human serum albumin (i.e., amino acids 1-585 of human serum albuminas shown in FIGS. 1 and 2 of EP Patent 0 322 094, which is hereinincorporated by reference in its entirety). In another preferredembodiment, polypeptides and/or antibodies of the present invention(including fragments or variants thereof) are fused with polypeptidefragments comprising, or alternatively consisting of, amino acidresidues 1-z of human serum albumin, where z is an integer from 369 to419, as described in U.S. Pat. No. 5,766,883 incorporated herein byreference in its entirety.

BSL1 (e.g., SEQ ID NO:4), BSL2 (e.g., SEQ ID NO:8, SEQ ID NO:132, or SEQID NO:134), or BSL3 (e.g., SEQ ID NO:16) polynucleotides encoding fusionproteins, and antibodies to these fusion proteins, are also encompassedby the invention. Such fusion proteins may, for example, facilitatepurification and may increase half-life in vivo. Antibodies fused orconjugated to the polypeptides of the present invention may also be usedin in vitro immunoassays and purification methods using methods known inthe art. See, e.g., Harbor et al., supra, and PCT publication WO93/21232; EP 439,095, Naramura et al. (1994) Immunol. Lett. 39:91-99;U.S. Pat. No. 5,474,981; Gillies et al. (1992) Proc. Natl. Acad. Sci.USA, 89:1428-1432; Fell et al. (1991) J. Immunol. 146:2446-2452, whichare incorporated by reference herein in their entireties. Antibodies toBSL1 (e.g., SEQ ID NO:5), BSL2 (e.g., SEQ ID NO:9, SEQ ID NO:133, or SEQID NO:135), or BSL3 (e.g., SEQ ID NO:17) fusion proteins can be used inany of the antibody-based methods for polypeptide identification,purification, and for antibody-format assays for diagnosis, treatment,and monitoring known in the art and/or disclosed herein.

The present invention further includes compositions comprising theB7-related polypeptides of the present invention fused or conjugated toantibody domains other than the variable region domain. For example, thepolypeptides of the present invention can be fused or conjugated to anantibody Fc region, or portion thereof. The antibody portion fused to apolypeptide of the present invention can comprise the constant region,hinge region, CH1 domain, CH2 domain, CH3 domain, or any combination ofwhole domains or portions thereof. The polypeptides can also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. (See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al. (1991) Proc.Natl. Acad. Sci. USA, 88:10535-10539; Zheng et al. (1995) J. Immunol.154:5590-5600; and Vil et al., Proc. Natl. Acad. Sci. USA,89:11337-11341, which are hereby incorporated by reference herein intheir entireties).

As discussed supra, the polypeptides corresponding to a polypeptide,polypeptide fragment, or a variant of one or more of a B7-related aminoacid sequence as set forth in FIGS. 1-6 can be fused or conjugated tothe above antibody portions to increase the in vivo half life of thepolypeptides, or for use in immunoassays using methods known in the art.Further, the polypeptides corresponding to one or more of the B7-relatedsequences as set forth in FIGS. 1-6 can be fused or conjugated to theabove antibody portions to facilitate purification. For guidance,chimeric proteins having the first two domains of the human CD4polypeptide and various domains of the constant regions of the heavy orlight chains of mammalian immunoglobulins have been described. (EP394,827; Traunecker et al. (1988) Nature, 331:84-86). The polypeptidesof the present invention fused or conjugated to an antibody, or portionthereof, having disulfide-linked dimeric structures (due to the IgG),for example, can also be more efficient in binding and neutralizingother molecules, than the monomeric secreted protein or protein fragmentalone. (Fountoulakis et al. (1995) J. Biochem. 270:3958-3964). In manycases, the Fc portion in a fusion protein is beneficial in therapy,diagnosis, and/or screening methods, and thus can result in, forexample, improved pharmacokinetic properties. (EP A 232, 262). In drugdiscovery, for example, human proteins, such as hIL-5, have been fusedwith Fc portions for the purpose of high-throughput screening assays toidentify antagonists of hIL-5. (See, Bennett et al. (1995) J. MolecularRecognition, 8:52-58; and Johanson et al. (1995) J. Biol. Chem.270:9459-9471). Alternatively, deleting the Fc portion after the fusionprotein has been expressed, detected, and purified, may be desired. Forexample, the Fc portion may hinder therapy and diagnosis if the fusionprotein is used as an antigen for immunizations.

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide, to facilitate theirpurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., Chatsworth, Calif.), among others, many of which arecommercially available. As described in Gentz et al. (1989) Proc. Natl.Acad. Sci. USA, 86:821-824, for instance, hexa histidine provides forconvenient purification of the fusion protein. Other peptide tags usefulfor purification include, but are not limited to, the “HA” tag, whichcorresponds to an epitope derived from the influenza hemagglutinin (HA)protein (Wilson et al. (1984) Cell, 37:767) and the “flag” tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure, forexample, to determine the efficacy of a given treatment regimen.Detection can be facilitated by coupling the antibody to a detectablesubstance. Nonlimiting examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive materials, positronemitting metals using various positron emission tomographies, andnonradioactive paramagnetic metal ions. The detectable substance can becoupled or conjugated either directly to the antibody (or fragmentthereof) or indirectly, through an intermediate (such as, for example, alinker known in the art) using techniques known in the art. (See, forexample, U.S. Pat. No. 4,741,900 for metal ions that can be conjugatedto antibodies for use as diagnostics according to the presentinvention).

Nonlimiting examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;nonlimiting examples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin; nonlimiting examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; a nonlimiting example of a luminescentmaterial includes luminol; nonlimiting examples of bioluminescentmaterials include luciferase, luciferin, and aequorin; and nonlimitingexamples of suitable radioactive material include iodine (¹²⁵I, ¹³¹I),carbon (¹⁴C), sulfur (3sus), tritium (³H); indium (¹¹¹In and otherradioactive isotopes of inidium); technetium (⁹⁹Tc, ^(99m)Tc), thallium(20′Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo),xenon (¹³³Xe), fluorine (¹⁹F), ¹⁵³Sm, ¹⁷⁷Lu, Gd, radioactive Pm,radioactive La, radioactive Yb, ¹⁶⁶Ho, ⁹⁰Y, radioactive Sc, radioactiveRe, radioactive Re, ¹⁴²Pr, ¹⁰⁵Rh, and ⁹⁷Ru.

In specific embodiments, the B7-related polypeptides of the inventionare attached to macrocyclic chelators useful for conjugating radiometalions, including, but not limited to, ¹¹¹In, ¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho, and¹⁵³Sm, to polypeptides. In a preferred embodiment, the radiometal ionassociated with the macrocyclic chelators attached to the B7-relatedpolypeptides of the invention is ¹¹¹In. In another preferred embodiment,the radiometal ion associated with the macrocyclic chelator attached tothe B7-related polypeptides of the invention is ⁹⁰Y. In specificembodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA). Inother specific embodiments, the DOTA is attached to the B7-relatedpolypeptides of the invention via a linker molecule.

Examples of linker molecules useful for conjugating DOTA to apolypeptide are commonly known in the art. (See, for example, DeNardo etal. (1998) Clin. Cancer Res. 4(10):2483-90; Peterson et al. (1999)Bioconjug. Chem. 10(4):553-557; and Zimmerman et al. (1999) Nucl. Med.Biol. 26(8): 943-950, which are hereby incorporated by reference intheir entirety. In addition, U.S. Pat. Nos. 5,652,361 and 5,756,065,which disclose chelating agents that can be conjugated to antibodies andmethods for making and using them, are hereby incorporated by referencein their entireties. Though U.S. Pat. Nos. 5,652,361 and 5,756,065 focuson conjugating chelating agents to antibodies, one skilled in the artcan readily adapt the methods disclosed therein in order to conjugatechelating agents to other polypeptides.

Antibodies can also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating therapeutic moieties to antibodies are wellknown, see, e.g., Arnon et al. (1985) “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, Monoclonal Antibodies AndCancer Therapy, Reisfeld et al., eds., Alan R. Liss, Inc., pp. 243-56;Hellstrom et al. (1987) “Antibodies For Drug Delivery”, Controlled DrugDelivery, 2nd Ed., Robinson et al. (eds.), Marcel Deldcer, Inc., pp.623-53; Thorpe, (1985) “Antibody Carriers Of Cytotoxic Agents In CancerTherapy: A Review”, Monoclonal Antibodies '84: Biological And ClinicalApplications, Pinchera et al., eds., pp. 475-506; Baldwin et al., eds.,(1985) “Analysis, Results, And Future Prospective Of The Therapeutic UseOf Radiolabeled Antibody In Cancer Therapy”, Monoclonal Antibodies ForCancer Detection And Therapy, Academic Press, pp. 303-316; and Thorpe etal. (1982) Immunol. Rev. 62:119-158. Alternatively, an antibody can beconjugated to a second antibody to form an antibody heteroconjugate,e.g., as described in U.S. Pat. No. 4,676,980 to Segal, which isincorporated herein by reference in its entirety. An antibody, i.e., anantibody specific for a B7-related polypeptide of this invention, withor without a therapeutic moiety conjugated to it, and administered aloneor in combination with cytotoxic factor(s) and/or cytokine(s), can beused as a therapeutic.

The antibodies of the invention can be utilized for immunophenotyping ofcell lines and biological samples. The translation product of the BSL1,BSL2, or BSL3-encoding sequences of the present invention can be usefulas cell specific marker(s), or more specifically, as cellular marker(s)that are differentially expressed at various stages of differentiationand/or maturation of particular cell types. Monoclonal antibodiesdirected against a specific epitope, or combination of epitopes, allowfor the screening of cellular populations expressing the marker. Varioustechniques utilizing monoclonal antibodies can be employed to screen forcellular populations expressing the marker(s), including magneticseparation using antibody-coated magnetic beads, “panning” withantibody(ies) attached to a solid matrix (i.e., tissue culture plate),and flow cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison etal. (1999) Cell, 96:737-749).

These techniques allow for the screening of particular populations ofcells, such as might be found with hematological malignancies (i.e.minimal residual disease (MRD) in acute leukemic patients) and“non-self” cells in transplantations to prevent Graft-versus-HostDisease (GVHD). Alternatively, these techniques allow for the screeningof hematopoietic stem and progenitor cells capable of undergoingproliferation and/or differentiation, as might be found in humanumbilical cord blood.

Antibodies according to this invention can be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include, but are not limited to, competitive and non-competitiveassay systems using techniques such as BIAcore analysis, FACS(Fluorescence Activated Cell Sorter) analysis, immunofluorescence,immunocytochemistry, Western blots, radioimmunoassays, ELISA (enzymelinked immunosorbent assays), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known and practiced in the art (see, e.g., Ausubel etal, eds., (1994) Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York, which is incorporated by reference hereinin its entirety). Nonlimiting, exemplary immunoassays are describedbriefly below.

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (i.e., 1% NP-40 or TritonX-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodiumphosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphataseand/or protease inhibitors (e.g. EDTA, PMSF, aprotinin, sodiumvanadate); adding the antibody of interest to the cell lysate;incubating for a period of time (e.g., 1 to 4 hr) at 4° C.; addingprotein A and/or protein G sepharose beads to the cell lysate;incubating for about 60 min or more at 4° C.; washing the beads in lysisbuffer; and resuspending the beads in SDS/sample buffer. The ability ofthe antibody of interest to immunoprecipitate a particular antigen canbe assessed by, for example, Western blot analysis. One of skill in theart would be knowledgeable as to the parameters that can be modified toincrease the binding of the antibody to an antigen and decrease thebackground (e.g., pre-clearing the cell lysate with sepharose beads).For further discussion regarding immunoprecipitation protocols, see,e.g., Ausubel et al, eds., (1994) Current Protocols in MolecularBiology, Vol. 1, John Wiley & Sons, Inc., New York, at 10.16.1.

Western blot analysis generally comprises preparing protein samples;electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS PAGE depending on the molecular weight of the antigen);transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon; blocking the membrane inblocking solution (e.g., PBS with 3% BSA or nonfat milk); washing themembrane in washing buffer (e.g., PBS-Tween®20); blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer; washing the membrane in washing buffer; blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., ³²P or ¹²⁵I) diluted in blocking buffer; washing the membrane inwash buffer; and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding Western blot protocols, see, e.g., Ausubelet al, eds. (1994) Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York, at 10.8.1.

ELISAs comprise preparing antigen; coating the wells of a 96 wellmicrotiter plate with antigen; adding to the wells the antibody ofinterest conjugated to a detectable compound such as an enzymaticsubstrate (e.g., horseradish peroxidase or alkaline phosphatase);incubating for a period of time; and detecting the presence of theantigen. In ELISAs, the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundcan be added to the wells. Further, instead of coating the wells withantigen, the antibody can be first coated onto the well. In this case, asecond antibody conjugated to a detectable compound can be added to theantibody-coated wells following the addition of the antigen of interest.One of skill in the art would be knowledgeable as to the parameters thatcan be modified to increase the signal detected, as well as othervariations of ELISAs known in the art.

In the initial steps, an ELISA assay may involve preparing an antibodyspecific to antigens of a B7-related polypeptide or peptide fragmentsthereof, preferably a monoclonal antibody. In addition, a reporterantibody can be used to recognize and bind to the monoclonal antibody.To the reporter antibody a detectable reagent may be attached, such as aradioactive isotope, a fluorescent moiety, or, in this example, anenzyme, such as horseradish peroxidase. To carry out an ELISA assay, asample can be removed from a host, e.g., a patient sample, and incubatedon a solid support, e.g., wells of a microtiter plate, or a polystyrenedish, to which the proteins in the sample can bind. Any free proteinbinding sites on the dish may then be blocked by incubating with anon-specific protein such as bovine serum albumin. The monoclonalantibody can then be added to the solid support, e.g., the wells or thedish, and allowed to incubate.

During the incubation time, the monoclonal antibodies may attach to anyB7-related polypeptides or peptides that have attached to thepolystyrene dish. All unbound monoclonal antibody can then be washedaway using an appropriate buffer solution. The reporter antibody, e.g.,linked to horseradish peroxidase, can be added to the support, therebyresulting in the binding of the reporter antibody to any monoclonalantibody that has bound to B7-related polypeptides or peptides that arepresent in the sample. Unattached reporter antibody can then be washedaway. Peroxidase substrate can be added to the support and the amount ofcolor developed in a given time period can be taken to provide ameasurement of the amount of B7-related polypeptides or peptides thatare present in a given volume of patient sample when compared against astandard curve. For further discussion regarding ELISAs, see, e.g.,Ausubel et al, eds. (1994) Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York, at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassayinvolving the incubation of labeled antigen (e.g., ³H or ¹²⁵I), or afragment or variant thereof, with the antibody of interest in thepresence of increasing amounts of labeled antigen, and the detection ofthe antibody bound to the labeled antigen. The affinity of the antibodyof interest for a B7-related polypeptide of the invention and thebinding off rates can be determined from the data by Scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, a B7-related polypeptide such asBSL1 (e.g., SEQ ID NO:2), BSL2 (e.g., SEQ ID NO:7, 11, or 13), or BSL3(e.g., SEQ ID NO:15) is incubated with antibody of interest conjugatedto a labeled compound (e.g., a compound labeled with ³H or ¹²⁵I) in thepresence of increasing amounts of an unlabeled second antibody. Thiskind of competitive assay between two antibodies, may also be used todetermine if two antibodies bind to the same or different epitopes.

In a preferred embodiment, BIAcore kinetic analysis is used to determinethe binding on and off rates of antibodies (including antibody fragmentsor variants thereof) to a B7-related polypeptide, or fragments orvariants of a B7-related polypeptide disclosed herein. Kinetic analysiscomprises analyzing the binding and dissociation of antibodies fromchips with immobilized B7-related polypeptide such as BSL1 (e.g., SEQ IDNO:2), BSL2 (e.g., SEQ ID NO:7, 11, or 13), or BSL3 (e.g., SEQ ID NO:15)on the chip surface.

Assays Utilizing B7-Related Nucleic Acids or Polypeptides

Expression analysis of B7-related factors: Several well-establishedtechniques can be used to determine the expression levels, patterns, andcell-type specificity of the B7-related factors. For example, mRNAlevels can be determined utilizing Northern blot analysis (J. C. Alwineet al. (1977) Proc. Natl. Acad. Sci. USA 74:5350-5354; I. M. Bird (1998)Methods Mol. Biol. 105:325-36.), whereby poly(A)⁺ RNA is isolated fromcells, separated by gel electrophoresis, blotted onto a support surface(e.g., nitrocellulose or Immobilon-Ny+ (Millipore Corp., Bedford,Mass.)), and incubated with a labeled (e.g., fluorescently labeled orradiolabeled) oligonucleotide probe that is capable of hybridizing withthe mRNA of interest. Alternatively, mRNA levels can be determined byquantitative (for review, see W. M. Freeman et al. (1999) Biotechniques26:112-122) or semi-quantitative RT-PCR analysis (Ren et al. Mol. BrainRes. 59:256-63). In accordance with this technique, poly(A)⁺ RNA isisolated from cells, used for cDNA synthesis, and the resultant cDNA isincubated with PCR primers that are capable of hybridizing with thetemplate and amplifying the template sequence to produce levels of thePCR product that are proportional to the cellular levels of the mRNA ofinterest. Another technique, in situ hybridization, can also be used todetermine mRNA levels (reviewed by A. K. Raap (1998) Mutat Res.400:287-298). In situ hybridization techniques allow the visualdetection of mRNA in a cell by incubating the cell with a labeled (e.g.,fluorescently labeled or digoxigenin labeled) oligonucleotide probe thathybridize to the mRNA of interest, and then examining the cell bymicroscopy.

Chromosomal mapping of B7-related genes: The chromosomal location ofB7-related genes can be determined by various techniques known in theart. For example, high-resolution chromosomal banding can be used(reviewed by M. Ronne (1990) In Vivo 4:337-65). High-resolution bandingtechniques utilize elongated chromosomes from cells at early mitoticstages, which have been synchronized using DNA-synthesis inhibitors(e.g., methotrexate or thymidine) or DNA-binding agents (e.g., ethidiumbromide). However, these techniques can only be used to map a gene to arelatively large region of a chromosome (˜3 Mb). For more accurate genemapping, fluorescence in situ hybridization (FISH) techniques can beused. In particular, high-resolution FISH techniques (A. Palotie et al.(1996) Ann. Med. 28:101-106) utilize free chromatin, DNA fibers, ormechanically-stretched chromosomes to map gene sequences ranging fromseveral kilobases to 300-kb in size. Alternatively, the chromosomallocation of a gene can be determined from the appropriate genomedatabase, for example, the Homo sapiens genome database available at theEntrez Genome website (National Center for Biotechnology Information,Bethesda, Md.).

Identification of T-cell ligands: The B7-related polypeptides orpeptides disclosed herein can be used to identify their cognate ligandson immune or inflammatory response cells, such as T-cells (i.e., CD28-or CTLA-4-related ligands). Candidate ligands, or fragments derivedtherefrom, can be identified and analyzed by many well-known methods inthe art (see T. E. Creighton, Ed. (1997) Proteins Structure: A PracticalApproach, IRL Press at Oxford Press, Oxford, England). For example,T-cell ligands that bind to the B7-related polypeptides or peptides canbe identified from extracts or lysates obtained from animal, preferablyhuman, immune or inflammatory response cells (e.g., T-cells). Theproteins obtained from these sources can be separated into bands usingsodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) andtransferred by electroblotting, for example, onto a suitable solid-phasesupport or membrane (e.g., nitrocellulose or polyvinylidene fluoride(PVDF)). The solid-phase support or membrane can then be incubated witha labeled form of a B7-related polypeptide or peptide, e.g., BSL1 (e.g.,SEQ ID NO:2), BSL2 (e.g., SEQ ID NO:7, 11, or 13), or BSL3 (e.g., SEQ IDNO:15). Bands that exhibit specific binding with the labeled B7-relatedpolypeptide or peptide can then be identified, isolated, purified, andanalyzed by amino acid analysis and/or Edman degradation to determinethe amino acid sequence of peptides derived therefrom.

As an alternative approach, a fusion protein comprising a B7-relatedpolypeptide can be attached to a solid support and incubated withextracts obtained from cells, such as CHO or COS cells, that aretransfected with an appropriate cDNA library. For example, a cDNAlibrary can be constructed from resting or activated immortal humanT-cell lines, such as CEM, HUT78, or Jurkat cell lines, or from restingor activated human T-cells derived from peripheral blood, tonsil,spleen, thymus or other specialized lymphoid tissues. Such cells can beactivated by the addition of anti-CD3 and anti-CD28 monoclonalantibodies, phytohemaglutinin (PHA), or phorbol 12-myristate-13-acetate(PMA) with ionomycin. The cDNA library construct can contain a removableepitope tag (see above) that is different from the fusion protein, andwill facilitate purification of the library expression product(s) thatassociate with the fusion protein. The isolated library expressionproduct(s) can then be isolated and characterized.

In addition, a fusion protein comprising a B7-related polypeptide can beattached to a solid support (e.g., a column comprising beads thatspecifically bind to the fusion protein) and incubated with lysatesobtained from cells, such as T-cells, that are enriched for integralmembrane proteins. The cellular proteins that associate with the fusionprotein can be isolated and then characterized using MALDI-TOF analysis(Matrix Assisted Laser Desorption Ionization Time Of Flight Analysis;reviewed by Yates J R 3rd. (1998) J. Mass Spectrom. 33:1-19; P. Chaurandet al. (1999) J. Am. Soc. Mass Spectrom. 10:91-103). Fusion proteins caninclude, for example, FLAG®—(B. L. Brizzard et al. (1994) Biotechniques16:730-735), 6×-HIS, and GST fusion proteins (see above), which can beattached to solid supports that are conjugated with anti-FLAG®antibodies, nickel, or glutathione molecules, respectively. Methods ofproducing and purifying such fusion proteins are well known in the art.

Another suitable ligand-binding assay is the yeast two-hybrid system(Fields et al. (1989) Nature 340:245-246; U.S. Pat. No. 5,283,173). Thetwo-hybrid system relies on the reconstitution of transcriptionactivation activity by association of the DNA-binding and transcriptionactivation domains of a transcriptional activator throughprotein-protein interaction. The yeast GAL4 transcriptional activatormay be used in this way, although other transcription factors have beenused and are well known in the art. To carryout the two-hybrid assay,the GAL4 DNA-binding domain and the GAL4 transcription activation domainare expressed, separately, as fusions to potential interactingpolypeptides. For example, one fusion protein can comprise a B7-relatedpolypeptide fused to the GAL4 DNA-binding domain. The other fusionprotein can comprise, for example, a T-cell cDNA library encodedpolypeptide fused to the GAL4 transcription activation domain. If thetwo, coexpressed fusion proteins interact in the nucleus of a host cell,a reporter gene (e.g. LacZ) is activated to produce a detectablephenotype. The host cells that show two-hybrid interactions can be usedto isolate the containing plasmids containing the cDNA librarysequences. These plasmids can be analyzed to determine the nucleic acidsequence and predicted polypeptide sequence of the candidate T-cellligand.

Related, in vivo, methods such as the three-hybrid (Licitra et al.(1996) Proc. Natl. Acad. Sci. USA 93:12817-12821), and reversetwo-hybrid (Vidal et al. (1996) Proc. Natl. Acad. Sci. USA93:10315-10320) systems may serve as alternative approaches.Commercially available two-hybrid systems such as the CLONTECHMatchmaker™ systems and protocols (CLONTECH, Palo Alto, Calif.) may bealso be used. (See also, A. R. Mendelsohn et al. (1994) Curr. Op:Biotech. 5:482; E. M. Phizicky et al. (1995) Microbiological Rev. 59:94;M. Yang et al. (1995) Nucleic Acids Res. 23:1152; S. Fields et al.(1994) Trends Genet. 10:286; and U.S. Pat. Nos. 6,283,173 and5,468,614).

Ligand sequence(s) obtained from ligand-binding assay(s) can be comparedwith subject sequences in available databases such as, withoutlimitation, GenPept, SWISS-PROT, and Incyte Genomics databases (IncyteGenomics). These databases, which contain previously identified andannotated sequences, may be searched for the full-length polypeptide andgene sequence using, for example, BLAST analysis (see above). In caseswhere the full-length sequences of the ligands are not available,extended or overlapping partial clones may be obtained by techniquesconventionally known and practiced in the art. Non-limiting examples ofsuch techniques include hybridization to plasmid or phage libraries ofgenomic DNA or cDNA; PCR from the same libraries using B7-related factorprimer pairs; or hybridization or PCR directly to genomic DNA or cDNA.These clones may then be sequenced and assembled into full-length genesusing the fragment sequence alignment program (PHRAP; Nickerson et al.(1997) Nucleic Acids Res. 25:2745-2751).

Assays for B7-related factor activity: Screening the fragments, mutantsor variants for those which retain characteristic B7-related polypeptideactivity as described herein can be accomplished using one or more ofseveral different assays. For example, appropriate cells, such as CHOcells, can be transfected with the cloned variants and then analyzed forcell surface phenotype by indirect immunofluorescence and flowcytometry. Cell surface expression of the transfected cells is evaluatedusing a monoclonal antibody specifically reactive with a cell surfaceform of a B7-related factor (see above). Production of secreted forms ofthe B7-related factors can be evaluated by immunoprecipitation using amonoclonal antibody specifically reactive with a B7-related factor.

Other, more preferred, assays take advantage of the functionalcharacteristics of the B7-related factors. As previously set forth, thebinding of the B7-related factors to its T-cell ligand(s) causes thecells to produce increased levels of lymphokines, particularly ofinterleukin-2. Thus, B7-related factor function can be assessed bymeasuring the synthesis of lymphokines, such as interleukin-2 or othernovel and as yet undefined cytokines, and/or assaying for T-cellproliferation by CD28⁺ T-cells that have received a primary activationsignal. Any one of several conventional assays for interleukin-2 can beemployed (see C. B. Thompson (1989) Proc. Natl. Acad. Sci. USA 86:1333).

The same basic functional assays can also be used to screen forB7-related polypeptides, peptides, fusion proteins, or antibodies thatblock T-cell activation. The ability of such proteins to block thenormal costimulatory signal and induce a state of anergy can bedetermined using subsequent attempts at stimulation of the T-cells withantigen presenting cells that express cell surface B cell activationantigen B7 and present antigen. If the T-cells are unresponsive to theactivation attempts, as determined by IL-2 synthesis and T-cellproliferation, a state of anergy has been induced and can be determinedby methods known in the art (see R. H. Schwartz (1990) Science248:1349-1356).

Modulators of B7-Related Factors

The BSL1, BSL2, and BSL3 polypeptides, polynucleotides, variants, orfragments thereof, can be used to screen for test agents (e.g.,agonists, antagonists, or inhibitors) that modulate the levels oractivity of the corresponding B7-related polypeptide. In addition,B7-related molecules can be used to identify endogenous modulators thatbind to BSL1, BSL2, or BSL3 polypeptides or polynucleotides in the cell.In one aspect of the present invention, the full-length BSL1 (e.g., SEQID NO:2), BSL2 (e.g., SEQ ID NO:7, 11, or 13), or BSL3 (e.g., SEQ IDNO:15) polypeptide is used to identify modulators. Alternatively,variants or fragments of a BSL1, BSL2, or BSL3 polypeptide are used.Such fragments may comprise, for example, one or more domains of theB7-related polypeptide (e.g., the extracellular and transmembranedomains) disclosed herein. Of particular interest are screening assaysthat identify agents that have relatively low levels of toxicity inhuman cells. A wide variety of assays may be used for this purpose,including in vitro protein-protein binding assays, electrophoreticmobility shift assays, immunoassays, and the like.

The term “modulator” as used herein describes any test agent, molecule,protein, peptide, or compound with the capability of directly orindirectly altering the physiological function, stability, or levels ofthe BSL1, BSL2, and BSL3 polypeptide. Modulators that bind to theB7-related polypeptides or polynucleotides of the invention arepotentially useful in diagnostic applications and/or pharmaceuticalcompositions, as described in detail herein. Test agents useful asmodulators may encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 50 and less than about 2,500 daltons. Suchmolecules can comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. Test agentswhich can be used as modulators often comprise cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups. Test agentscan also comprise biomolecules including peptides, saccharides, fattyacids, steroids, purines, pyrimidines, derivatives, structural analogs,or combinations thereof.

Test agents finding use as modulators may include, for example, 1)peptides such as soluble peptides, including Ig-tailed fusion peptidesand members of random peptide libraries (see, e.g., Lam et al. (1991)Nature 354:82-84; Houghten et al. (1991) Nature 354:84-86) andcombinatorial chemistry-derived molecular libraries made of D- and/orL-configuration amino acids; 2) phosphopeptides (e.g., members of randomand partially degenerate, directed phosphopeptide libraries, see, e.g.,Songyang et al., (1993) Cell 72:767-778); 3) antibodies (e.g.,polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and singlechain antibodies as well as Fab, F(ab′)₂, Fab expression libraryfragments, and epitope-binding fragments of antibodies); and 4) smallorganic and inorganic molecules.

Test agents and modulators can be obtained from a wide variety ofsources including libraries of synthetic or natural compounds. Syntheticcompound libraries are commercially available from, for example,Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton,N.J.), Brandon Associates (Merrimack, N.H.), and Microsource (NewMilford, Conn.). A rare chemical library is available from AldrichChemical Company, Inc. (Milwaukee, Wis.). Natural compound librariescomprising bacterial, fungal, plant or animal extracts are availablefrom, for example, Pan Laboratories (Bothell, Wash.). In addition,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides.

Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts can be readily produced. Methods forthe synthesis of molecular libraries are readily available (see, e.g.,DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909; Erb et al.(1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994) J.Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carell et al.(1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew.Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem.37:1233). In addition, natural or synthetic compound libraries andcompounds can be readily modified through conventional chemical,physical and biochemical means (see, e.g., Blondelle et al. (1996)Trends in Biotech. 14:60), and may be used to produce combinatoriallibraries. In another approach, previously identified pharmacologicalagents can be subjected to directed or random chemical modifications,such as acylation, alkylation, esterification, amidification, and theanalogs can be screened for BSL1-, BSL2-, and BSL3-modulating activity.

Numerous methods for producing combinatorial libraries are known in theart, including those involving biological libraries; spatiallyaddressable parallel solid phase or solution phase libraries; syntheticlibrary methods requiring deconvolution; the ‘one-bead one-compound’library method; and synthetic library methods using affinitychromatography selection. The biological library approach is limited topolypeptide libraries, while the other four approaches are applicable topolypeptide, non-peptide oligomer, or small molecule libraries ofcompounds (K. S. Lam (1997) Anticancer Drug Des. 12:145).

Libraries may be screened in solution (e.g., Houghten, (1992)Biotechniques 13:412-421), or on beads (Lam, (1991) Nature 354:82-84),chips (Fodor, (1993) Nature 364:555-556), bacteria or spores (LadnerU.S. Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad.Sci. USA 89:1865-1869), or on phage (Scott and Smith, (1990) Science249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990)Proc. Natl. Acad. Sci. USA 97:6378-6382; Felici (1991) J. Mol. Biol.222:301-310; Ladner, supra).

Where the screening assay is a binding assay, a BSL1, BSL2, or BSL3polypeptide, fusion protein, polynucleotide, analog, or fragmentthereof, may be joined to a label, where the label can directly orindirectly provide a detectable signal. Various labels includeradioisotopes, fluorescers, chemiluminescers, enzymes, specific bindingmolecules, particles, e.g. magnetic particles, and the like. Specificbinding molecules include pairs, such as biotin and streptavidin,digoxin and antidigoxin, etc. For the specific binding members, thecomplementary member would normally be labeled with a molecule thatprovides for detection, in accordance with known procedures.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc., that are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc., may be used. Thecomponents are added in any order that produces the requisite binding.Incubations are performed at any temperature that facilitates optimalactivity, typically between 4° and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high-throughput screening. Normally, between 0.1 and 1 hour willbe sufficient. In general, a plurality of assay mixtures is run inparallel with different agent concentrations to obtain a differentialresponse to these concentrations. Typically, one of these concentrationsserves as a negative control, i.e. at zero concentration or below thelevel of detection.

To perform cell-free screening assays, it may be desirable to immobilizeeither a BSL1, BSL2, or BSL3 polypeptide, polynucleotide, or fragment toa surface to facilitate identification of modulators that bind to thesemolecules, as well as to accommodate automation of the assay. Forexample, a fusion protein comprising a BSL1 (e.g., SEQ ID NO:2), BSL2(e.g., SEQ ID NO:7, 11, or 13), or BSL3 (e.g., SEQ ID NO:15) polypeptideand an affinity-tag can be produced as described in detail herein. Inone embodiment, a GST-fusion protein comprising a BSL1, BSL2, or BSL3polypeptide is adsorbed onto glutathione sepharose beads (SigmaChemical, St. Louis, Mo.) or glutathione-derivatized microtiter plates.Cell lysates (e.g., containing ³⁵S-labeled polypeptides) are added tothe polypeptide-coated beads under conditions to allow complex formation(e.g., at physiological conditions for salt and pH). Followingincubation, the polypeptide-coated beads are washed to remove anyunbound polypeptides, and the amount of immobilized radiolabel isdetermined. Alternatively, the complex is dissociated and the radiolabelpresent in the supernatant is determined. In another approach, the beadsare analyzed by SDS-PAGE to identify BSL1-, BSL2-, or BSL3-bindingpolypeptides. Various binding assays can be used to identify agonist orantagonists that alter the function or levels of a BSL1 (e.g., SEQ IDNO:2), BSL2 (e.g., SEQ ID NO:7, 11, or 13), or BSL3 (e.g., SEQ ID NO:15)polypeptide. Such assays are designed to detect the interaction of testagents with BSL1, BSL2, or BSL3 polypeptides, polynucleotides,functional equivalents, or fragments thereof. Interactions may bedetected by direct measurement of binding. Alternatively, interactionsmay be detected by indirect indicators of binding, such asstabilization/destabilization of protein structure, oractivation/inhibition of biological function. Non-limiting examples ofuseful binding assays are detailed below.

Modulators that bind to BSL1, BSL2, or BSL3 polypeptides,polynucleotides, functional equivalents, or fragments thereof, can beidentified using real-time Bimolecular Interaction Analysis (BIA;Sjolander et al. (1991) Anal. Chem. 63:2338-2345; Szabo et al. (1995)Curr. Opin. Struct. Biol. 5:699-705; e.g., BIAcore™; LKB Pharmacia,Sweden). Modulators can also be identified by scintillation proximityassays (SPA, described in U.S. Pat. No. 4,568,649). Binding assays usingmitochondrial targeting signals (Hurt et al. (1985) EMBO J. 4:2061-2068;Eilers and Schatz, (1986) Nature 322:228-231) a plurality of definedpolymers synthesized on a solid substrate (Fodor et al. (1991) Science251:767-773) may also be employed.

Two-hybrid systems may be used to identify modulators (see, e.g., U.S.Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al.(1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;and Brent WO 94/10300). Alternatively, three-hybrid (Licitra et al.(1996) Proc. Natl. Acad. Sci. USA 93:12817-12821), and reversetwo-hybrid (Vidal et al. (1996) Proc. Natl. Acad. Sci. USA93:10315-10320) systems may be used. Commercially available two-hybridsystems such as the CLONTECH Matchmaker™ systems and protocols (CLONTECHLaboratories, Inc., Palo Alto, Calif.) are also useful (see also, A. R.Mendelsohn et al. (1994) Curr. Op. Biotech. 5:482; E. M. Phizicky et al.(1995) Microbiological Rev. 59:94; M. Yang et al. (1995) Nucleic AcidsRes. 23:1152; S. Fields et al. (1994) Trends Genet. 10:286; and U.S.Pat. Nos. 6,283,173 and 5,468,614).

Several methods of automated assays have been developed in recent yearsso as to permit screening of tens of thousands of test agents in a shortperiod of time. High-throughput screening methods are particularlypreferred for use with the present invention. The binding assaysdescribed herein can be adapted for high-throughput screens, oralternative screens may be employed. For example, continuous format highthroughput screens (CF-HTS) using at least one porous matrix allows theresearcher to test large numbers of test agents for a wide range ofbiological or biochemical activity (see U.S. Pat. No. 5,976,813 toBeutel et al.). Moreover, CF-HTS can be used to perform multi-stepassays.

Diagnostics

According to another embodiment of the present invention, the B7-relatedpolynucleotides, or fragments thereof, may be used for diagnosticpurposes. The B7-related polynucleotides that may be used includeoligonucleotide sequences, complementary RNA and DNA molecules, andPNAs. BSL1 (e.g., SEQ ID NO:1 or 3), BSL2 (e.g., SEQ ID NO:6, 10, 12, or131), and BSL3 (e.g., SEQ ID NO:14) polynucleotides, or fragmentsthereof, can be used to quantitate levels of BSL1, BSL2, or BSL3 mRNA inbiological samples in which expression (or under- or overexpression) ofBSL1, BSL2, and BSL3 polynucleotide may be correlated with disease. Thediagnostic assay may be used to distinguish between the absence,presence, increase, and decrease of the expression of BSL1, BSL2, andBSL3, and to monitor regulation of BSL1, BSL2, and BSL3 polynucleotidelevels during therapeutic treatment or intervention.

In one aspect, PCR probes can be used to detect B7-relatedpolynucleotide sequences, including BSL1, BSL2, and BSL3 genomic DNAsequences and BSL1-, BSL2-, and BSL3-related nucleic acid sequences. Thespecificity of the probe, whether it is made from a highly specificregion, e.g., at least 8 to 10 or 12 or 15 contiguous nucleotides in the5′ regulatory region, or a less specific region, e.g., especially in the3′ coding region, and the stringency of the hybridization oramplification (maximal, high, intermediate, or low) will determinewhether the probe identifies only naturally occurring sequences encodingthe B7-related polypeptide, alleles thereof, or related sequences.

Probes may also be used for the detection of BSL1-, BSL2-, andBSL3-related sequences, and should preferably contain at least 60%,preferably greater than 90%, identity to a BSL1 (e.g., SEQ ID NO:1 or3), BSL2 (e.g., SEQ ID NO:6, 10, 12, or 131), and BSL3 (e.g., SEQ IDNO:14) polynucleotide, or a complementary sequence, or fragmentsthereof. The probes of this invention may be DNA or RNA, the probes maycomprise all or a fragment of the nucleotide sequence of BSL1 (e.g., SEQID NO:2), BSL2 (e.g., SEQ ID NO:7, 11, or 13), or BSL3 (e.g., SEQ IDNO:15), or a complementary sequence thereof, and may include promoter,enhancer elements, and introns of the naturally occurring BSL1, BSL2, orBSL3 polynucleotide.

Methods for producing specific probes for B7-related polynucleotidesinclude the cloning of nucleic acid sequences of BSL1 (e.g., SEQ IDNO:2), BSL2 (e.g., SEQ ID NO:7, 11, or 13), or BSL3 (e.g., SEQ IDNO:15), or a fragment thereof, into vectors for the production of mRNAprobes. Such vectors are known in the art, commercially available, andmay be used to synthesize RNA probes in vitro by means of the additionof the appropriate RNA polymerases and the appropriate labelednucleotides. Hybridization probes may be labeled by a variety ofdetector/reporter groups, e.g., radionucleotides such as ³²P or ³⁵S, orenzymatic labels, such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems, and the like.

A wide variety of labels and conjugation techniques are known andemployed by those skilled in the art and may be used in various nucleicacid and amino acid assays. Means for producing labeled hybridization orPCR probes for detecting sequences related to polynucleotides encoding aBSL1, BSL2, or BSL3 polypeptide include oligo-labeling, nicktranslation, end-labeling, or PCR amplification using a labelednucleotide. Alternatively, BSL1, BSL2, or BSL3 polynucleotide sequences,or any portions or fragments thereof, may be cloned into a vector forthe production of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase, such as T7, T3, orSP(6) and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (e.g., from Amersham PharmaciaBiotech, Inc., Piscataway, N.J.; Promega Corp., Madison Wis.; and U.S.Biochemical Corp., U.S. Biochemical Amersham, Cleveland, Ohio). Suitablereporter molecules or labels which may be used include radionucleotides,enzymes, fluorescent, chemiluminescent, or chromogenic agents, as wellas substrates, cofactors, inhibitors, magnetic particles, and the like.

B7-related polynucleotide sequences, or fragments, or complementarysequences thereof, can be used in Southern or Northern analysis, dotblot, or other membrane-based technologies; in PCR technologies; or indip stick, pin, ELISA or biochip assays utilizing fluids or tissues frompatient biopsies to detect the status of, e.g., levels or overexpressionof BSL1, BSL2, or BSL3, or to detect altered BSL1, BSL2, or BSL3expression. Such qualitative or quantitative methods are well known inthe art (G. H. Keller and M. M. Manak (1993) DNA Probes, 2^(nd) Ed,Macmillan Publishers Ltd., England; D. W. Dieffenbach and G. S. Dveksler(1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y.; B. D. Hames and S. J. Higgins (1985) Gene Probes 1, 2,IRL Press at Oxford University Press, Oxford, England).

BSL1, BSL2, and BSL3 oligonucleotides may be chemically synthesized,generated enzymatically, or produced from a recombinant source.Oligomers will preferably comprise two nucleotide sequences, one with asense orientation (5′→3′) and another with an antisense orientation(3′→5′), employed under optimized conditions for identification of aspecific gene or condition. The same two oligomers, nested sets ofoligomers, or even a degenerate pool of oligomers may be employed underless stringent conditions for detection and/or quantification of closelyrelated DNA or RNA sequences.

Methods suitable for quantifying the expression of B7-related factorsinclude radiolabeling or biotinylating nucleotides, co-amplification ofa control nucleic acid, and standard curves onto which the experimentalresults are interpolated (P. C. Melby et al. (1993) J. Immunol. Methods159:235-244; and C. Duplaa et al. (1993) Anal. Biochem. 229-236). Thespeed of quantifying multiple samples may be accelerated by running theassay in an ELISA format where the oligomer of interest is presented invarious dilutions and a spectrophotometric or colorimetric responsegives rapid quantification.

In a particular aspect, a nucleic acid sequence complementary to aB7-related polynucleotide, or fragment thereof, may be useful in assaysthat detect diseases relating to aberrant immune responses, particularlythose described herein. A BSL1, BSL2, and/or BSL3 polynucleotide can belabeled by standard methods, and added to a biological sample from asubject under conditions suitable for the formation of hybridizationcomplexes. After a suitable incubation period, the sample can be washedand the signal is quantified and compared with a standard value. If theamount of signal in the test sample is significantly altered from thatof a comparable negative control (normal) sample, the altered levels ofBSL1, BSL2, and/or BSL3 nucleotide sequence can be correlated with thepresence of the associated disease. Such assays may also be used toevaluate the efficacy of a particular prophylactic or therapeuticregimen in animal studies, in clinical trials, or for an individualpatient.

To provide a basis for the diagnosis of a disease associated withaltered expression of one or more B7-related factors, a normal orstandard profile for expression is established. This may be accomplishedby incubating biological samples taken from normal subjects, eitheranimal or human, with a sequence complementary to a BSL1, BSL2, BSL3polynucleotide, or a fragment thereof, under conditions suitable forhybridization or amplification. Standard hybridization may be quantifiedby comparing the values obtained from normal subjects with those from anexperiment where a known amount of a substantially purifiedpolynucleotide is used. Standard values obtained from normal samples maybe compared with values obtained from samples from patients who aresymptomatic for the disease. Deviation between standard and subject(patient) values is used to establish the presence of the condition.

Once the disease is diagnosed and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in a normal individual. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

With respect to diseases involving a hyperactive or hypoactive immuneresponse, the presence of an abnormal levels (decreased or increased) ofB7-related transcript in a biological sample (e.g., body fluid, cells,tissues, or cell or tissue extracts) from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier, thereby preventing the development or furtherprogression of the disease.

In one particular aspect, BSL1, BSL2, and BSL3 oligonucleotides may beused for PCR-based diagnostics. For example, PCR can be used to performGenetic Bit Analysis (GBA) of BSL1, BSL2, and/or BSL3 in accordance withpublished methods (T. T. Nikiforov et al. (1994) Nucleic Acids Res.22(20):4167-75; T. T. Nikiforov et al. (1994) PCR Methods Appl.3(5):285-91). In PCR-based GBA, specific fragments of genomic DNAcontaining the polymorphic site(s) are first amplified by PCR using oneunmodified and one phosphorothioate-modified primer. The double-strandedPCR product is rendered single-stranded and then hybridized toimmobilized oligonucleotide primer in wells of a multi-well plate.Notably, the primer is designed to anneal immediately adjacent to thepolymorphic site of interest. The 3′ end of the primer is extended usinga mixture of individually labeled dideoxynucleoside triphosphates. Thelabel on the extended base is then determined. Preferably, GBA isperformed using semi-automated ELISA or biochip formats (see, e.g., S.R. Head et al. (1997) Nucleic Acids Res. 25(24):5065-71; T. T. Nikiforovet al. (1994) Nucleic Acids Res. 22(20):4167-75).

In another embodiment of the present invention, oligonucleotides, orlonger fragments derived from at least one B7-related polynucleotidesequence described herein may be used as targets in a microarray (e.g.,biochip) system. The microarray can be used to monitor the expressionlevel of large numbers of genes simultaneously (to produce a transcriptimage), and to identify genetic variants, mutations, and polymorphisms.This information may be used to determine gene function, to understandthe genetic basis of a disease, to diagnose disease, and to develop andmonitor the activities of therapeutic or prophylactic agents.Preparation and use of microarrays have been described in WO 95/11995 toChee et al.; D. J. Lockhart et al. (1996) Nature Biotechnology14:1675-1680; M. Schena et al. (1996) Proc. Natl. Acad. Sci. USA93:10614-10619; U.S. Pat. No. 6,015,702 to P. Lal et al.; J. Worley etal. (2000) Microarray Biochip Technology, M. Schena, ed., BiotechniquesBook, Natick, Mass., pp. 65-86; Y. H. Rogers et al. (1999) Anal.Biochem. 266(1):23-30; S. R. Head et al. (1999) Mol. Cell. Probes.13(2):81-7; S. J. Watson et al. (2000) Biol. Psychiatry 48(12):1147-56.

In one application of the present invention, microarrays containingarrays of B7-related polynucleotide sequences can be used to measure theexpression levels of B7-related factors in an individual. In particular,to diagnose an individual with a condition or disease correlated withaltered BSL1, BSL2, and/or BSL3 expression levels, a sample from a humanor animal (containing, e.g., mRNA) can be used as a probe on a biochipcontaining an array of BSL1, BSL2, and/or BSL3 polynucleotides (e.g.,DNA) in decreasing concentrations (e.g., 1 ng, 0.1 ng, 0.01 ng, etc.).The test sample can be compared to samples from diseased and normalsamples. Biochips can also be used to identify BSL1, BSL2, and BSL3mutations or polymorphisms in a population, including but not limitedto, deletions, insertions, and mismatches. For example, mutations can beidentified by: (i) placing B7-related polynucleotides of this inventiononto a biochip; (ii) taking a test sample (containing, e.g., mRNA) andadding the sample to the biochip; (iii) determining if the test sampleshybridize to the B7-related polynucleotides attached to the chip undervarious hybridization conditions (see, e.g., V. R. Chechetkin et al.(2000) J. Biomol. Struct Dyn. 18(1):83-101). Alternatively microarraysequencing can be performed (see, e.g., E. P. Diamandis (2000) Clin.Chem. 46(10):1523-5).

In another embodiment of this invention, a B7-related nucleic acidsequence, or a complementary sequence, or fragment thereof, can be usedas probes which are useful for mapping the naturally occurring genomicsequence. The sequences may be mapped to a particular chromosome, to aspecific region of a chromosome, or to artificial chromosomeconstructions (HACs), yeast artificial chromosomes (YACs), bacterialartificial chromosomes (BACs), bacterial PI constructions, or singlechromosome cDNA libraries (see C. M. Price (1993) Blood Rev., 7:127-134and by B. J. Trask (1991) Trends Genet 7:149-154).

In a further embodiment of the present invention, antibodies whichspecifically bind to a BSL1, BSL2, or BSL3 polypeptide may be used forthe diagnosis of conditions or diseases characterized by underexpressionor overexpression of a BSL1, BSL2, or BSL3 polynucleotide orpolypeptide, or in assays to monitor patients being treated with a BSL1,BSL2, or BSL3 polypeptide, peptide, or fusion protein, or a BSL1, BSL2,or BSL3 agonist, antagonist, or inhibitor. The antibodies useful fordiagnostic purposes may be prepared in the same manner as those for usein therapeutic methods, described herein. Diagnostic assays for a BSL1,BSL2, or BSL3 polypeptide include methods that utilize the antibody anda label to detect the protein in biological samples (e.g., human bodyfluids, cells, tissues, or extracts of cells or tissues). The antibodiesmay be used with or without modification, and may be labeled by joiningthem, either covalently or non-covalently, with a reporter molecule. Awide variety of reporter molecules that are known in the art may beused, several of which are described herein.

A number of fluorescent materials are known and can be utilized to labela B7-related polypeptide or antibodies that specifically bind thereto.These include, for example, fluorescein, rhodamine, auramine, Texas Red,AMCA blue and Lucifer Yellow. A particular detecting material isanti-rabbit antibody prepared in goats and conjugated with fluoresceinthrough an isothiocyanate. B7-related polypeptides or antibodies theretocan also be labeled with a radioactive element or with an enzyme. Theradioactive label can be detected by any of the currently availablecounting procedures. Preferred isotopes include ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl,⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re. Enzyme labels arelikewise useful, and can be detected by any of the presently utilizedcalorimetric, spectrophotometric, fluorospectrophotometric,amperometric, or gasometric techniques. The enzyme can be conjugated byreaction with bridging molecules such as carbodiimides, diisocyanates,glutaraldehyde, and the like. Many enzymes, which can be used in theseprocedures, are known and can be utilized. Preferred are peroxidase,β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucoseoxidase plus peroxidase, and alkaline phosphatase (see, e.g., U.S. Pat.Nos. 3,654,090; 3,850,752; and 4,016,043).

Antibody-based diagnostics and their application are familiar to thoseskilled in the art and may be used in accordance with the presentinvention. As non-limiting examples, “competitive” (U.S. Pat. Nos.3,654,090 and 3,850,752), “sandwich” (U.S. Pat. No. 4,016,043), and“double antibody,” or “DASP” assays may be used. Several proceduresincluding ELISA, RIA, and FACS for measuring B7-related polypeptidelevels are known in the art and provide a basis for diagnosing alteredor abnormal levels of B7-related polypeptide expression. Normal orstandard values for B7-related polypeptide expression are established byincubating biological samples taken from normal subjects, preferablyhuman, with antibody to the B7-related polypeptide under conditionssuitable for complex formation. The amount of standard complex formationmay be quantified by various methods; photometric means are preferred.Levels of the B7-related polypeptide expressed in the subject sample,negative control (normal) sample, and positive control (disease) sampleare compared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

In another of its aspects, this invention relates to diagnostic kits fordetecting B7-related polynucleotide(s) or polypeptide(s) as it relatesto a disease or susceptibility to a disease, particularly the disordersof the immune system described herein. Such kits comprise one or more ofthe following: (a) a B7-related polynucleotide, preferably thenucleotide sequence of BSL1 (e.g., SEQ ID NO:1 or 3), BSL2 (e.g., SEQ IDNO:6, 10, 12, or 131), or BSL3 (e.g., SEQ ID NO:14), or a fragmentthereof; or (b) a nucleotide sequence complementary to that of (a); or(c) a B7-related polypeptide, preferably the polypeptide of BSL1 (e.g.,SEQ ID NO:2), BSL2 (e.g., SEQ ID NO:7, 11, or 13), or BSL3 (e.g., SEQ IDNO:15), or a fragment thereof; or (d) an antibody to a B7-relatedpolypeptide, preferably to the polypeptide of BSL1 (e.g., SEQ ID NO:2),BSL2 (e.g., SEQ ID NO:7, 11, or 13), or BSL3 (e.g., SEQ ID NO:15), or anantibody bindable fragment thereof. It will be appreciated that in anysuch kits, (a), (b), (c), or (d) may comprise a substantial componentand that instructions for use can be included. The kits may also containperipheral reagents such as buffers, stabilizers, etc.

The present invention also includes a test kit for genetic screeningthat can be utilized to identify mutations in B7-related factors. Byidentifying patients with mutated BSL1, BSL2, and/or BSL2 DNA andcomparing the mutation to a database that contains known mutations inBSL1, BSL2, and BSL3, and a particular condition or disease,identification and/or confirmation of, a particular condition or diseasecan be made. Accordingly, such a kit would comprise a PCR-based testthat would involve transcribing the patients mRNA with a specificprimer, and amplifying the resulting cDNA using another set of primers.The amplified product would be detectable by gel electrophoresis andcould be compared with known standards for BSL1, BSL2, and/or BSL3.Preferably, this kit would utilize a patient's blood, serum, or salivasample, and the DNA would be extracted using standard techniques.Primers flanking a known mutation would then be used to amplify afragment of BSL1, BSL2, and/or BSL3. The amplified piece would then besequenced to determine the presence of a mutation.

Therapeutics

Pharmaceutical compositions: The present invention contemplatescompositions comprising a B7-related nucleic acid, polypeptide, fusionprotein, antibody, ligand, modulator (e.g., agonist, antagonist, orinhibitor), or fragments or functional variants thereof, and aphysiologically acceptable carrier, excipient, or diluent as describedin detail herein. The present invention further contemplatespharmaceutical compositions useful in practicing the therapeutic methodsof this invention. Preferably, a pharmaceutical composition includes, inadmixture, a pharmaceutically acceptable excipient (carrier) and one ormore of a B7-related polypeptide, fusion protein, nucleic acid, ligand,modulator, antibody, or fragment or functional equivalent thereof, asdescribed herein, as an active ingredient. Because B7-relatedpolypeptides or peptides are naturally occurring cellular components,they may be administered to an individual's circulatory system withminimal risk of undesired immunological complications.

The preparation of pharmaceutical compositions that contain biologicalreagents as active ingredients is well understood in the art. Typically,such compositions are prepared as injectables, either as liquidsolutions or suspensions, however, solid forms suitable for solution in,or suspension in, liquid prior to injection can also be prepared. Thepreparation can also be emulsified. The active therapeutic ingredient isoften mixed with excipients that are pharmaceutically acceptable andcompatible with the active ingredient. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol, or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH-buffering agents, which enhance the effectivenessof the active ingredient.

Pharmaceutical compositions can be produced and employed in treatmentprotocols according to established methods depending on the disorder ordisease to be treated (see, for example, P. D. Mayne (1996) ClinicalChemistry in Diagnosis and Treatment, 6^(th) ed., Oxford UniversityPress, Oxford, England; Gilman et al., Eds. (1990) Goodman and Gilman's:The Pharmacological Basis of Therapeutics, 8th ed., Pergamon Press; Aviset al., Eds. (1993) Pharmaceutical Dosage Forms: Parenteral Medications,Dekker, New York, N.Y.; and Lieberman et al., Eds. (1990) PharmaceuticalDosage Forms: Disperse Systems, Dekker, New York, N.Y.).

Pharmaceutical compositions may be produced as neutral or salt forms.Salts can be formed with many acids, including, but not limited to,hydrochloric, sulfuric, acetic, lactic, tartaric, malic and succinicacids. Compositions can take the form of solutions, suspensions,suppositories, tablets, pills, capsules, sustained release compounds, orpowders. Such formulations can contain 10%-95% (w/w) of the activeingredient, preferably 25%-70% (w/w). If the active compound isadministered by injection, for example, about 1 μg-3 mg and preferablyfrom about 20 μg-500 μg of active compound (e.g., B7-related fusionprotein or antibody) per dosage unit may be administered. Pharmaceuticalpreparations and compositions can also contain one or morephysiologically acceptable carrier(s), excipient(s), diluent(s),disintegrant(s), lubricant(s), plasticizer(s), filler(s), colorant(s),dosage vehicle(s), absorption enhancer(s), stabilizer(s), orbacteriocide(s). The production and formulation of such compositions andpreparations are carried out by methods known and practiced in the art.

Exemplary formulations are given below:

Intravenous Formulation I: Ingredient mg/ml BSL1, BSL2, or BSL3 MAb 5.0dextrose USP 45.0 sodium bisulfite USP 3.2 edetate disodium USP 0.1water for injection q.s.a.d. 1.0 ml

Intravenous Formulation II: Ingredient mg/ml BSL1, BSL2, or BSL3 MAb 5.0sodium bisulfite USP 3.2 disodium edetate USP 0.1 water for injectionq.s.a.d. 1.0 ml

Intravenous Formulation III Ingredient mg/ml BSL1, BSL2, or BSL3protein, Ig-fusion protein, or agonist 10.0 sodium bisulfite USP 3.2disodium edetate USP 0.1 water for injection q.s.a.d. 1.0 ml

Intravenous Formulation IV Ingredient mg/ml BSL1, BSL 2, or BSL3protein, Ig-fusion protein, or agonist 10.0 dextrose USP 45.0 sodiumbisulfite USP 3.2 edetate disodium USP 0.1 water for injection q.s.a.d.1.0 ml

As used herein, “pg” means picogram, “ng” means nanogram, “μg” meanmicrogram, “mg” means milligram, “μl” mean microliter, “ml” meansmilliliter, and “l” means liter.

Following the preparation of pharmaceutical compositions, they may beplaced in appropriate containers and labeled for the treatment ofindicated conditions. Such labeling can include amount, frequency, andmethod of administration. Preparations may be administered systemicallyby oral or parenteral routes. Non-limiting parenteral routes ofadministration include subcutaneous, intramuscular, intraperitoneal,intravenous, transdermal, inhalation, intranasal, intra-arterial,intrathecal, enteral, sublingual, or rectal administration.

A therapeutically effective amount of a pharmaceutical compositioncontaining one or more B7-related polypeptides, fusion proteins, peptidefragments, or antibodies that specifically react with these componentsis an amount sufficient to reduce, ameliorate, or eliminate a disease ordisorder related to altered activation levels of immune or inflammatoryresponse cells, such as T-cells. An effective amount can be introducedin one administration or over repeated administrations to an individualbeing treated. Therapeutic administration can be followed byprophylactic administration, after treatment of the disease. Aprophylactically effective amount is an amount effective to preventdisease and will depend upon the specific illness and subject. Thetherapeutically effective dose may be estimated initially, for example,either in cell culture assays or in animal models, usually mice, rats,rabbits, dogs, sheep, goats, pigs, or non-human primates. The animalmodel may also be used to determine the maximum tolerated dose andappropriate route of administration. Such information can then be usedto determine useful doses and routes for administration in humans.

Administration of the therapeutic compositions of the present inventionto a subject can be carried out using known procedures, at dosages andfor periods of time effective to achieve the desired result. Forexample, a therapeutically active amount of B7-related polypeptides,fusion proteins, peptides, or antibodies that react with thesecomponents may vary according to factors such as the age, sex, andweight of the individual, and the ability of the treatment to elicit adesired response in the individual. Dosages may be adjusted to providethe optimum therapeutic response. For example, several sequential dosesmay be administered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

Gene transfer therapy: In addition, host cells that are geneticallyengineered to carry the gene encoding a B7-related polypeptide, fusionprotein, or peptide fragment comprising a fragment of a BSL1 (e.g., SEQID NO:2), BSL2 (e.g., SEQ ID NO:7, 11, or 13), or BSL3 (e.g., SEQ IDNO:15) polypeptide sequence, can be introduced into an individual inneed of immunomodulation. Following expression and production of theB7-related polypeptide or peptide by the host cell, the so-producedB7-related polypeptide, fusion protein, or peptide can act to bindCD28/CTLA-4 and/or CD28-/CTLA-4-related ligand(s) to modulate theactivation of immune or inflammatory response cells (e.g., T-cells) inthe recipient. Host cells may be genetically engineered by a variety ofmolecular techniques and methods known to those having skill in the art,for example, transfection, infection, or transduction. Transduction asused herein commonly refers to cells that have been geneticallyengineered to contain a foreign or heterologous gene via theintroduction of a viral or non-viral vector into the cells. Transfectionmore commonly refers to cells that have been genetically engineered tocontain a foreign gene harbored in a plasmid, or non-viral vector. Hostcells can be transfected or transduced by different vectors and thus canserve as gene delivery vehicles to transfer the expressed products intomuscle.

Although viral vectors are preferred for gene transfer therapies, cellscan be genetically engineered to contain nucleic acid sequences encodingthe desired gene product(s) by various methods known in the art. Forexample, cells can be genetically engineered by fusion, transfection,lipofection mediated by the use of liposomes, electroporation,precipitation with DEAE-Dextran or calcium phosphate, particlebombardment (biolistics) with nucleic acid-coated particles (e.g., goldparticles), microinjection, or genetically engineered microorganisms (K.Yazawa et al. (2000) Cancer Gene Ther. 7:269-274). Vectors forintroducing heterologous (i.e., foreign) nucleic acid (DNA or RNA) intomuscle cells for the expression of active bioactive products are wellknown in the art. Such vectors possess a promoter sequence, preferably apromoter that is cell-specific and placed upstream of the sequence to beexpressed. The vectors may also contain, optionally, one or moreexpressible marker genes for expression as an indication of successfultransfection and expression of the nucleic acid sequences contained inthe vector. In addition, vectors can be optimized to minimize undesiredimmunogenicity and maximize long-term expression of the desired geneproduct(s) (see Nabel (1999) Proc. Natl. Acad. Sci. USA 96:324-326).Moreover, vectors can be chosen based on cell-type that is targeted fortreatment. For example, vectors for the treatment of tumor or cancercells have been described (P. L. Hallenbeck et al. (1999) Hum. GeneTher. 10:1721-1733; T. Shibata et al. (2000) Gene Ther. 7:493-498; M.Puhlmann et al. (2000) Cancer Gene Ther. 7:66-73; N. Krauzewicz et al.(2000) Adv. Exp. Med. Biol. 465:73-82).

Illustrative examples of vehicles or vector constructs for transfectionor infection of the host cells include replication-defective viralvectors, DNA virus or RNA virus (retrovirus) vectors, such asadenovirus, herpes simplex virus and adeno-associated viral vectors.Adeno-associated virus vectors are single stranded and allow theefficient delivery of multiple copies of nucleic acid to the cell'snucleus. Preferred are adenovirus vectors. The vectors will normally besubstantially free of any prokaryotic DNA and may comprise a number ofdifferent functional nucleic acid sequences. An example of suchfunctional sequences may be a DNA region comprising transcriptional andtranslational initiation and termination regulatory sequences, includingpromoters (e.g., strong promoters, inducible promoters, and the like)and enhancers which are active in the host cells. Also included as partof the functional sequences is an open reading frame (polynucleotidesequence) encoding a protein of interest. Flanking sequences may also beincluded for site-directed integration. In some situations, the5′-flanking sequence will allow homologous recombination, thus changingthe nature of the transcriptional initiation region, so as to providefor inducible or noninducible transcription to increase or decrease thelevel of transcription, as an example.

In general, the encoded and expressed B7-related factor may beintracellular, i.e., retained in the cytoplasm, nucleus, or an organelleof a cell, or may be secreted by the cell. For secretion, the naturalsignal sequence present in the B7-related structural gene may beretained. When the polypeptide or peptide is a fragment of a B7-relatedfactor that is larger, a signal sequence may be provided so that, uponsecretion and processing at the processing site, the desired proteinwill have the natural sequence. Specific examples of coding sequences ofinterest for use in accordance with the present invention include theBSL1 (e.g., SEQ ID NO:2), BSL2 (e.g., SEQ ID NO:7, 11, or 13), or BSL3(e.g., SEQ ID NO:15) polypeptide coding sequences. As previouslymentioned, a marker may be present for selection of cells containing thevector construct. The marker may be an inducible or non-inducible geneand will generally allow for positive selection under induction, orwithout induction, respectively. Examples of marker genes includeneomycin, dihydrofolate reductase, glutamine synthetase, and the like.

The vector employed will generally also include an origin of replicationand other genes that are necessary for replication in the host cells, asroutinely employed by those having skill in the art. As an example, thereplication system comprising the origin of replication and any proteinsassociated with replication encoded by a particular virus may beincluded as part of the construct. The replication system must beselected so that the genes encoding products necessary for replicationdo not ultimately transform the cells. Such replication systems arerepresented by replication-defective adenovirus (see G. Acsadi et al.(1994) Hum. Mol. Genet. 3:579-584) and by Epstein-Barr virus. Examplesof replication defective vectors, particularly, retroviral vectors thatare replication defective, are BAG, (see Price et al. (1987) Proc. Natl.Acad. Sci. USA, 84:156; Sanes et al. (1986) EMBO J., 5:3133). It will beunderstood that the final gene construct may contain one or more genesof interest, for example, a gene encoding a bioactive metabolicmolecule. In addition, cDNA, synthetically produced DNA or chromosomalDNA may be employed utilizing methods and protocols known and practicedby those having skill in the art.

According to one approach for gene therapy, a vector encoding aB7-related factor is directly injected into the recipient cells (in vivogene therapy). Alternatively, cells from the intended recipients areexplanted, genetically modified to encode a B7-related factor, andreimplanted into the donor (ex vivo gene therapy). An ex vivo approachprovides the advantage of efficient viral gene transfer, which issuperior to in vivo gene transfer approaches. In accordance with ex vivogene therapy, the host cells are first infected with engineered viralvectors containing at least one B7-related gene encoding a B7-relatedgene product, suspended in a physiologically acceptable carrier orexcipient such as saline or phosphate buffered saline, and the like, andthen administered to the host. The desired gene product is expressed bythe injected cells, which thus introduce the gene product into the host.The introduced gene products can thereby be utilized to treat orameliorate a disorder that is related to altered levels of theactivation of immune or inflammatory response cells (e.g., T-cells).

Methods of immunomodulation: In accordance with the present invention,the BSL1, BSL2, and BSL3 nucleic acid and polypeptide sequences can beused in the development of therapeutic reagents having the ability toeither up-regulate (amplify) or down-regulate (suppress) immuneresponses (e.g., T-cell activation). In particular, B7-relatedpolypeptides may interact with CD28 and thereby up-regulate immune cellactivity. Alternatively, B7-related polypeptides may interact withCTLA-4 and thereby down-regulate immune cell activity. For example,soluble, dimeric forms of the B7-related polypeptides that bind to theCD28 and/or CD28-related ligand(s) but fail to provide a costimulatorysignal to T-cells, can be used to block T-cell activation, and therebyprovide a specific means by which to induce tolerance in a subject.Similarly, antibodies directed against one or more B7-related factorscan be used to block the interaction between the B7-related factors andtheir cognate ligand(s), thereby preventing the activation of immune orinflammatory response cells (e.g., T-cells). In addition, fusionproteins comprising at least a fragment of a B7-related factor fused toat least the Fc domain of an IgG molecule can be used to up- ordown-regulate cells expressing the cognate ligand(s) of the B7-relatedfactor (e.g., T-cells). Furthermore, antisense or triplexoligonucleotides that bind to the nucleotide sequence of one or moreB7-related factors can be used to decrease the expression these factors.In contrast, cell surface, multivalent forms of B7-related factors thatbind to CD28 and/or CD28-related ligand(s) and provide a costimulatorysignal to immune or inflammatory response cells, such as T-cells, can beused to increase the activation of these cells. It is also possible toutilize more than one B7-related polypeptide, fusion protein, antibody,or therapeutically active fragments thereof, in order to up-regulate ordown-regulate the activity of immune or inflammatory cells (e.g.,T-cells) in an animal or human subject.

In addition, it may also be advantageous to employ B7-relatedtherapeutics in conjunction with other therapeutics, surgeries, ortreatments. For example, pharmaceutical compositions comprisingBSL2vcvc-Ig or anti-BLS2 MAbs may be co-administered with one or moreimmunosuppressants including, but not limited to, corticosteroids suchas cortisone, hydrocortisone (e.g., Cortef®), prednisone (e.g.,Deltasone®, Meticorten®, or Orasone®), prednisolone (e.g.,Delta-Cortef®, Pediapred®, or Prelone®), triamcinolone (e.g.,Aristocort® or Kenacort®), methylprednisolone (e.g., Medrol®),dexamethasone (e.g., Decadron®, Dexone®, or Hexadrol®), andbetamethasone (e.g., Celestone®); and other drugs including tacrolimus(e.g., Prograf® or FK506); azathioprine (e.g., Imuran); methotrexate(e.g., Rheumatrex®); glatiramer acetate (e.g., Copaxone®); cladribine(Leustatin); cyclophosphamide (e.g., Endoxan®, Cytoxan®, or Neosar®);Roquinimex (e.g., Linomide®); mitoxantrone (e.g., Novantrone®);mycophenolate mofetil (e.g., Cellcept®); cyclosporine (e.g., cyclosporinA; Sandimmune®); rapamycin (FRAP/mTOR inhibitor; sirolimus, e.g.,Rapamune®); antithymocyte antibodies, for example, lymphocyte immuneglobulin (Atgam®), anti-Tac, and Rh(D) immune globulin (e.g., Rhogam orGamulin); and similar drugs. In contrast, pharmaceutical compositionscomprising BSL3-Ig may be co-administered with one or moreimmunostimulants, including, but not limited to, Bacille Calmette-Guerin(BCG), Levamisole, intravenous immune globulin (IVIG); cytokines such asinterferon-α, interferon-γ, interferon-β-1b, IL-2 (e.g., recombinanthuman IL-2), G-CSF, and GM-CSF, and similar drugs.

Given the structure and function of the B7-related factors disclosedherein, it is possible to up-regulate or down-regulate the function of aB7-related factor in a number of ways. Down-regulating or preventing oneor more B7-related factor functions (i.e., preventing high levellymphokine synthesis by activated T-cells) should be useful in treatingautoimmune diseases, such as rheumatoid arthritis, multiple sclerosis,Lupus erythematosus, Hashimoto's thyroiditis, primary mixedema, Graves'disease, pernicious anemia, autoimmune atrophic gastritis, insulindependent diabetes mellitus, good pasture's syndrome, myasthenia gravis,pemphigus, Crohn's disease, sympathetic opthalmia, autoimmune uveitis,autoimmune hemolytic anemis, idiopathic thrombocytopenia, primarybiliary cirrhosis, ulcerative colitis, Sjogren's syndrome, polymyositisand mixed connective tissue disease. B7-related factors may also bedown-regulated for the treatment of inflammation related to psoriasis,chronic obstructive pulmonary disease, asthma, and atherosclerosis. Inaddition, B7-related factors may be down-regulated for the treatment oftissue, bone marrow, and organ transplantation, and graft versus hostdisease. For example, blockage of T-cell function should result inreduced tissue destruction in tissue transplantation. Typically, intissue transplants, rejection of the transplant is initiated by itsrecognition as foreign material, followed by an immune reaction thatdestroys the transplant. The B7-related molecules of the presentinvention can also be used to treat or prevent cancers as described indetail below.

The B7-related nucleic acid molecules provided by the present inventioncan be used to design therapeutics to block the function of one or moreB7-related factors. In particular, antisense or triplex oligonucleotidescan be administered to prevent the expression of the BSL1, BSL2, and/orBSL3 factors. For example, an oligonucleotide (e.g., DNAoligonucleotide) that hybridizes to a BSL1, BSL2, and/or BSL3 mRNA canbe used to target the mRNA for RnaseH digestion. Alternatively, anoligonucleotide that hybridizes to the translation initiation site of aBSL1 (e.g., SEQ ID NO:1 or 3), BSL2 (e.g., SEQ ID NO:6, 10, 12, or 131),or BSL3 (e.g., SEQ ID NO:14) mRNA be used to prevent translation of themRNA. In another approach, oligonucleotides that bind to thedouble-stranded DNA of the BSL1, BSL2, and/or BSL3 gene(s) can beadministered. Such oligonucleotides can form a triplex construct andprevent the unwinding and transcription of the DNA encoding the targetedB7-related factor. In all cases, the appropriate oligonucleotide can besynthesized, formulated as a pharmaceutical composition, andadministered to a subject. The synthesis and utilization of antisenseand triplex oligonucleotides have been previously described (e.g., H.Simon et al. (1999) Antisense Nucleic Acid Drug Dev. 9:527-31; F. X.Barre et al. (2000) Proc. Natl. Acad. Sci. USA 97:3084-3088; R. Elez etal. (2000) Biochem. Biophys. Res. Commun. 269:352-6; E. R. Sauter et al.(2000) Clin. Cancer Res. 6:654-60).

In the context of this invention, antisense oligonucleotides arenaturally-occurring oligonucleotide species or synthetic species formedfrom naturally-occurring subunits or their close homologues. Antisenseoligonucleotides may also include moieties that function similarly tooligonucleotides, but have non-naturally-occurring portions. Thus,antisense oligonucleotides may have altered sugar moieties orinter-sugar linkages. Exemplary among these are phosphorothioate andother sulfur containing species which are known in the art.

In preferred embodiments, at least one of the phosphodiester bonds ofthe antisense oligonucleotide has been substituted with a structure thatfunctions to enhance the ability of the compositions to penetrate intothe region of cells where the RNA whose activity is to be modulated islocated. It is preferred that such substitutions comprisephosphorothioate bonds, methyl phosphonate bonds, or short chain alkylor cycloalkyl structures. In accordance with other preferredembodiments, the phosphodiester bonds are substituted with structureswhich are, at once, substantially non-ionic and non-chiral, or withstructures which are chiral and enantiomerically specific. Persons ofordinary skill in the art will be able to select other linkages for usein the practice of the invention.

Antisense oligonucleotides may also include species that include atleast some modified base forms. Thus, purines and pyrimidines other thanthose normally found in nature may be so employed. Similarly,modifications on the furanosyl portions of the nucleotide subunits mayalso be effected, as long as the essential tenets of this invention areadhered to. Examples of such modifications are 2′-O-alkyl- and2′-halogen-substituted nucleotides. Some non-limiting examples ofmodifications at the 2′ position of sugar moieties which are useful inthe present invention include OH, SH, SCH₃, F, OCH₃, OCN, O(CH₂)_(n)NH₂and O(CH₂)_(n)CH₃, where n is from 1 to about 10. Such antisenseoligonucleotides are functionally interchangeable with naturaloligonucleotides or synthesized oligonucleotides, which have one or moredifferences from the natural structure. All such analogs arecomprehended by this invention so long as they function effectively tohybridize with BSL1, BSL2, or BSL3 DNA or RNA to inhibit the functionthereof.

For antisense therapeutics, the oligonucleotides in accordance with thisinvention preferably comprise from about 3 to about 50 subunits. It ismore preferred that such oligonucleotides and analogs comprise fromabout 8 to about 25 subunits and still more preferred to have from about12 to about 20 subunits. As defined herein, a “subunit” is a base andsugar combination suitably bound to adjacent subunits throughphosphodiester or other bonds.

Antisense oligonulcleotides can be produced by standard techniques (see,e.g., Shewmaker et al., U.S. Pat. No. 5,107,065). The oligonucleotidesused in accordance with this invention may be conveniently and routinelymade through the well-known technique of solid phase synthesis.Equipment for such synthesis is available from several vendors,including PE Applied Biosystems (Foster City, Calif.). Any other meansfor such synthesis may also be employed, however, the actual synthesisof the oligonucleotides is well within the abilities of thepractitioner. It is also will known to prepare other oligonucleotidesuch as phosphorothioates and alkylated derivatives.

The oligonucleotides of this invention are designed to be hybridizablewith BSL1, BSL2, or BSL3 RNA (e.g., mRNA) or DNA. For example; anoligonucleotide (e.g., DNA oligonucleotide) that hybridizes toB7-related mRNA can be used to target the mRNA for RnaseH digestionAlternatively, an oligonucleotide that hybridizes to the translationinitiation site of B7-related mRNA can be used to prevent translation ofthe mRNA. In another approach, oligonucleotides that bind to thedouble-stranded DNA of BSL1, BSL2, or BSL3 can be administered. Sucholigonucleotides can form a triplex construct and inhibit thetranscription of the DNA encoding BSL1, BSL2, or BSL3 polypeptides.Triple helix pairing prevents the double helix from opening sufficientlyto allow the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described (see, e.g., J. E. Gee et al. (1994) Molecular andImmunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.).

As non-limiting examples, antisense oligonucleotides may be targeted tohybridize to the following regions: mRNA cap region; translationinitiation site; translational termination site; transcriptioninitiation site; transcription termination site; polyadenylation signal;3′ untranslated region; 5′ untranslated region; 5′ coding region; midcoding region; and 3′ coding region. Preferably, the complementaryoligonucleotide is designed to hybridize to the most unique 5′ sequencein BSL1, BSL2, or BSL3, including any of about 15-35 nucleotidesspanning the 5′ coding sequence. Appropriate oligonucleotides can bedesigned using OLIGO software (Molecular Biology Insights, Inc.,Cascade, Colo.).

In accordance with the present invention, the antisense oligonucleotidecan be synthesized, formulated as a pharmaceutical composition, andadministered to a subject. The synthesis and utilization of antisenseand triplex oligonucleotides have been previously described (e.g., H.Simon et al. (1999) Antisense Nucleic Acid Drug Dev. 9:527-31; F. X.Barre et al. (2000) Proc. Natl. Acad. Sci. USA 97:3084-3088; R. Elez etal. (2000) Biochem. Biophys. Res. Commun. 269:352-6; E. R. Sauter et al.(2000) Clin. Cancer Res. 6:654-60). Alternatively, expression vectorsderived from retroviruses, adenovirus, herpes or vaccinia viruses, orfrom various bacterial plasmids may be used for delivery of nucleotidesequences to the targeted organ, tissue or cell population. Methodswhich are well known to those skilled in the art can be used toconstruct recombinant vectors which will express nucleic acid sequencethat is complementary to the nucleic acid sequence encoding a BSL1,BSL2, or BSL3 polypeptide. These techniques are described both inSambrook et al. (1989) and in Ausubel et al. (1992). For example, BSL1,BSL2, or BSL3 expression can be inhibited by transforming a cell ortissue with an expression vector that expresses high levels ofuntranslatable sense or antisense sequences. Even in the absence ofintegration into the DNA, such vectors may continue to transcribe RNAmolecules until they are disabled by endogenous nucleases. Transientexpression may last for a month or more with a non-replicating vector,and even longer if appropriate replication elements included in thevector system.

Various assays may be used to test the ability of specific antisenseoligonucleotides to inhibit BSL1, BSL2, or BSL3 expression. For example,mRNA levels can be assessed Northern blot analysis (Sambrook et al.(1989); Ausubel et al. (1992); J. C. Alwine et al. (1977) Proc. Natl.Acad. Sci. USA 74:5350-5354; I. M. Bird (1998) Methods Mol. Biol.105:325-36), quantitative or semi-quantitative RT-PCR analysis (see,e.g., W. M. Freeman et al. (1999) Biotechniques 26:112-122; Ren et al.(1998) Mol. Brain Res. 59:256-63; J. M. Cale et al. (1998), Methods Mol.Biol. 105:351-71), or in situ hybridization (reviewed by A. K. Raap(1998) Mutat. Res. 400:287-298). Alternatively, antisenseoligonucleotides may be assessed by measuring levels of BSL1, BSL2, orBSL3 polypeptide, e.g., by western blot analysis, indirectimmunofluorescence, immunoprecipitation techniques (see, e.g., J. M.Walker (1998) Protein Protocols on CD-ROM, Humana Press, Totowa, N.J.).

The B7-related polypeptide sequences provided by the present inventionmay also be useful in the design of therapeutic agents to block orenhance the activity of immune response cells (e.g., T-cells). Forexample, a fusion protein comprising the soluble portion of a B7-relatedpolypeptide conjugated with the Fc domain of human IgG can beconstructed by standard recombinant techniques, described above. TheBSL1-Ig (e.g., SEQ ID NO:5), BSL2-Ig (e.g., SEQ ID NO:9, SEQ ID NO:133,or SEQ ID NO:135), and/or BSL3-Ig (e.g., SEQ ID NO:17), fusion proteinscan be prepared as a pharmaceutical composition and administered to asubject. The BSL1-Ig, BSL2-Ig, and/or BSL3-Ig fusion proteins can beused to target specific T-cells for destruction, thereby reducingoverall T-cell activation. Such treatment methods can be modeled onanimal experiments, which utilize CTLA-4-Ig to prevent cardiac allograftrejection (Turka et al., supra). It will be understood by a personskilled in the art that such methods may be adapted for use in humans,and for use with other conditions, including various transplants andautoimmune diseases. Alternatively, certain BSL1-Ig, BSL2-Ig, and/orBSL3-Ig fusion proteins can be used to enhance T-cell activation. Forexample, BSL3-Ig fusion proteins can be used as co-stimulatory moleculesas disclosed in detail herein.

As an alternative approach, antibodies that specifically react withB7-related polypeptides or peptides can be used to block the activity ofimmune or inflammatory response cells (e.g., T-cells). Antibodies orrelated antibody fragments that bind to peptides or polypeptidescomprising the BSL1 (e.g., SEQ ID NO:2), BSL2 (e.g., SEQ ID NO:7, 11, or13), or BSL3 (e.g., SEQ ID NO:15) sequences can be formulated aspharmaceutical compositions and administered alone or in combination toa subject. Such antibodies can then inhibit the interaction of theB7-related polypeptides with CD28 and/or CD28-related ligands, andthereby prevent T-cell activation. Treatments utilizing antibodiesdirected against B7-related factors may be modeled on animalexperiments, which use antibodies against CD28, B7-1, or B7-2 (D. J.Lenshow et al. (1995) Transplantation 60:1171-1178; Y. Seko et al.(1998) Circ. Res. 83:463-469; A. Haczku et al. (1999) Am. J. Respir.Crit. Care Med. 159:1638-1643). One skilled in the art may adapt suchmethods for use in humans; and for use with various conditions involvinginflammation or transplantation. It is noted that antibody-basedtherapeutics produced from non-human sources can cause an undesiredimmune response in human subjects. To minimize this problem, chimericantibody derivatives can be produced. Chimeric antibodies combine anon-human animal variable region with a human constant region. Chimericantibodies can be constructed according to methods known in the art (seeMorrison et al. (1985) Proc. Natl. Acad. Sci. USA 81:6851; Takeda et al.(1985) Nature 314:452; U.S. Pat. No. 4,816,567 of Cabilly et al.; U.S.Pat. No. 4,816,397 of Boss et al.; European Patent Publication EP171496; EP 0173494; United Kingdom Patent GB 2177096B). In addition,antibodies can be further “humanized” by any of the techniques known inthe art, (e.g., Teng et al. (1983) Proc. Natl. Acad. Sci. USA80:7308-7312; Kozbor et al. (1983) Immunology Today 4: 7279; Olsson etal. (1982) Meth. Enzymol. 92:3-16; International Patent Application No.WO 92/06193; EP 0239400). Humanized antibodies can be also be obtainedfrom commercial sources (e.g., Scotgen Limited, Middlesex, GreatBritain). Immunotherapy with a humanized antibody may result inincreased long-term effectiveness for the treatment of chronic diseasesituations or situations requiring repeated antibody treatments.

In yet another approach, an isolated ligand of a B7-related factor canbe used to down-regulate the activity of immune or inflammatory responsecells (e.g., T-cells). For example, a soluble fusion protein comprisinga B7-related factor ligand can be produced, isolated, and used toproduce a pharmaceutical composition in accordance with the methodsdescribed in detail herein. This pharmaceutical composition can then beadministered to a subject to bind to one or more endogenous B7-relatedfactor(s) and block the activation of immune or inflammatory responsecells (e.g., T-cells) as previously described.

Up-regulation of a B7-related factor function may also be useful intherapy. Because viral infections are cleared primarily by cytotoxicT-cells, an increase in cytotoxic activity would be therapeuticallyuseful in situations where more rapid or thorough clearance of aninfective viral agent would be beneficial to an animal or human subject.Notably, B7-1 acts to increase the cytotoxicity of T-cells thoughinteractions with its cognate ligand(s). Thus, soluble active forms ofB7-related polypeptides can be administered for the treatment of localor systemic viral infections, such as immunodeficiency (e.g., HIV),papilloma (e.g., HPV), herpes (e.g., HSV), encephalitis, influenza(e.g., human influenza virus A), and common cold (e.g., humanrhinovirus) viral infections. For example, pharmaceutical formulationsof active multivalent B7-related factors can be administered topicallyto treat viral skin diseases such as herpes lesions or shingles, orgenital warts. Alternatively, pharmaceutical compositions of active,multivalent B7-related factors can be administered systemically to treatsystemic viral diseases such as AIDS, influenza, the common cold, orencephalitis.

In addition, modulation of B7-related factor function may be useful inthe induction of tumor immunity. For example, tumor cells (e.g.,sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, or carcinomacells) can be genetically engineered to carry a nucleic acid encoding atleast a fragment of at least one B7-related factor, such as BSL1 (e.g.,SEQ ID NO:2), BSL2 (e.g., SEQ ID NO:7, 11, or 13), or BSL3 (e.g., SEQ IDNO:15), and then administered to a subject to traverse tumor-specifictolerance in the subject. Notably, ectopic expression of B7-1 in B7negative murine tumor cells has been shown to induce T-cell mediatedspecific immunity accompanied by tumor rejection and prolongedprotection to tumor challenge in mice (L. Chen et al., supra; S.Townsend et al., supra; S. Baskar et al., supra). Tumor or cancer cellgene therapy treatments utilizing B7-related factors may be modeled onanimal experiments (see K. Dunussi-Joannopoulos et al. (1997) J.Pediatr. Hematol. Oncol. 19:356-340; K. Hiroishi et al. (1999) GeneTher. 6:1988-1994; B. K. Martin et al. (1999) J. Immunol. 162:6663-6670;M. Kuiper et al. (2000) Adv. Exp. Med. Biol. 465:381-390), or humanphase I trial experiments (H. L. Kaufman et al. (2000) Hum. Gene Ther.11:1065-1082), which use B7-1 or B7-2 for gene transfer therapy. It willbe understood that such methods may be adapted for use with varioustumor or cancer cells. Additionally, tumor immunity may be achieved byadministration of a B7-related fusion protein that directly stimulatesthe immune cells (see e.g., International Patent Application No. WO01/21796 to V. Ling et al.).

The experiments described herein indicate that BSL2-4616811-Ig(BSL2vcvc-Ig) fusion protein can be used alone or in conjunction withone or more anti-BSL2 MAbs for therapeutic applications. BSL2vcvcligand(s) and MAbs against BSL2 ligand(s) may also be useful astherapeutics. In particular, BSL2vcvc-Ig fusion protein (e.g., SEQ IDNO:9) can be used to inhibit disease progression in any disease whereexcessive or inappropriate activation of T-cells plays an importantrole. Such diseases would include, for example, acute and chronictransplant rejection, rheumatoid arthritis, multiple sclerosis,psoriasis, or other diseases described in detail herein. In addition,BSL2vcvc-Ig may also be used for specific applications such asxenotransplantation.

The experiments described herein demonstrate that anti-BSL2 MAbs (e.g.,anti-BSL2-1 MAb, anti-BSL2-2 MAb, anti-BSL2-3 MAb, anti-BLS2-4 MAb, andanti-BSL2-5 MAb) function synergistically with BSL2-4616811-Ig(BSL2vcvc-Ig) to inhibit T-cell proliferation. This indicates thatanti-BSL2 MAbs may be used alone as therapeutics, if endogenous BSL2vcvcis expressed in sufficient amount by the subject's cells. Ifinsufficient endogenous BSL2vcvc is expressed, co-administration of antiBSL2 MAbs with BSL2vcvc-Ig may be more effective than administration ofeither alone. In certain cases, however, it may be desirable toadminister either BSL2-4616811-Ig (BSL2vcvc-Ig) or anti-BSL2 MAbsseparately.

It may also be possible to engineer a bi-specific monoclonal antibodythat could bring together endogenous BSL2vcvc and endogenous BSL2vcvcligand on T-cells. The bi-specific antibody may thereby mimic the effectof co-administration of BSL2-4616811-Ig (BSL2vcvc-Ig) and one or moreanti-BSL2 MAbs. In addition, signaling MAbs raised-against BSL2vcvcligand may be used to mimic the effect of BSL2vcvc-Ig, whereas blockingMAbs raised against BSL2vcvc ligand may act as immunostimulatoryfactors. It is also possible that soluble BSL2vcvc ligand may be used asan immunostimulatory factor.

Pharmacogenetics: The B7-related polypeptides and polynucleotides of thepresent invention are also useful in pharmacogenetic analysis, i.e., thestudy of the relationship between an individual's genotype and thatindividual's response to a therapeutic composition or drug. See, e.g.,Eichelbaum, M. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985,and Linder, M. W. (1997) Clin. Chem. 43(2):254-266. The genotype of theindividual can determine the way a therapeutic acts on the body or theway the body metabolizes the therapeutic. Further, the activity of drugmetabolizing enzymes affects both the intensity and duration oftherapeutic activity. Differences in the activity or metabolism oftherapeutics can lead to severe toxicity or therapeutic failure.Accordingly, a physician or clinician may consider applying knowledgeobtained in relevant pharmacogenetic studies in determining whether toadminister a B7-related polypeptide, polynucleotide, functionalequivalent, fragment, or modulator, as well as tailoring the dosageand/or therapeutic or prophylactic treatment regimen with a B7-relatedpolypeptide, polynucleotide, functional equivalent, fragment, ormodulator.

In general, two types of pharmacogenetic conditions can bedifferentiated. Genetic conditions can be due to a single factor thatalters the way the drug act on the body (altered drug action), or afactor that alters the way the body metabolizes the drug (altered drugmetabolism). These conditions can occur either as rare genetic defectsor as naturally occurring polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy which results in haemolysis after ingestion ofoxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans)and consumption of fava beans.

The discovery of genetic polymorphisms of drug metabolizing enzymes(e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6and CYP2C19) has provided an explanation as to why some patients do notobtain the expected drug effects or show exaggerated drug response andserious toxicity after taking the standard and safe dose of a drug.These polymorphisms are expressed in two phenotypes in the population,the extensive metabolizer (EM) and poor metabolizer (PM). The prevalenceof PM is different among different populations. The gene coding forCYP2D6 is highly polymorphic and several mutations have been identifiedin PM, which all lead to the absence of functional CYP2D6. Poormetabolizers quite frequently experience exaggerated drug response andside effects when they receive standard doses. If a metabolite is theactive therapeutic moiety, PM show no therapeutic response. This hasbeen demonstrated for the analgesic effect of codeine mediated by itsCYP2D6-formed metabolite morphine. At the other extreme, ultra-rapidmetabolizers fail to respond to standard doses. Recent studies havedetermined that ultra-rapid metabolism is attributable to CYP2D6 geneamplification.

By analogy, genetic polymorphism or mutation may lead to allelicvariants of BSL1, BSL2, and/or BSL3 in the population which havedifferent levels of activity. The BSL1, BSL2, and/or BSL3 polypeptidesor polynucleotides thereby allow a clinician to ascertain a geneticpredisposition that can affect treatment modality. Thus, in a BSL-basedtreatment, polymorphism or mutation may give rise to individuals thatare more or less responsive to treatment. Accordingly, dosage wouldnecessarily be modified to maximize the therapeutic effect within agiven population containing the polymorphism. As an alternative togenotyping, specific polymorphic polypeptides or polynucleotides can beidentified.

To identify genes that predict drug response, several pharmacogeneticmethods can be used. One pharmacogenomics approach, “genome-wideassociation”, relies primarily on a high-resolution map of the humangenome. This high-resolution map shows previously identifiedgene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants). A high-resolution genetic mapcan then be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, a high-resolution map can be generatedfrom a combination of some 10 million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Inthis way, treatment regimens can be tailored to groups of geneticallysimilar individuals, taking into account traits that may be common amongsuch genetically similar individuals. See, e.g., D. R. Pfost et al.(2000) Trends Biotechnol. 18(8):334-8.

As another example, the “candidate gene approach”, can be used.According to this method, if a gene that encodes a drug target is known,all common variants of that gene can be fairly easily identified in thepopulation and it can be determined if having one version of the geneversus another is associated with a particular drug response.

As yet another example, a “gene expression profiling approach”, can beused. This method involves testing the gene expression of an animaltreated with a drug (e.g., a B7-related polypeptide, polynucleotide,functional equivalent, fragment, or modulator) to determine whether genepathways related to toxicity have been turned on.

Information obtained from one of the pharmacogenetics approachesdescribed herein can be used to determine appropriate dosage andtreatment regimens for prophylactic or therapeutic treatment anindividual. This knowledge, when applied to dosing or drug selection;can avoid adverse reactions or therapeutic failure and thus enhancetherapeutic or prophylactic efficiency when treating a subject with aB7-related polypeptide, polynucleotide, functional equivalent, fragment,or modulator.

B7-related polypeptides or polynucleotides are also useful formonitoring therapeutic effects during clinical trials and othertreatment. Thus, the therapeutic effectiveness of an agent that isdesigned to increase or decrease gene expression, polypeptide levels, oractivity can be monitored over the course of treatment using theB7-related polypeptides or polynucleotides. For example, monitoring canbe performed by: (i) obtaining a pre-administration sample from asubject prior to administration of the agent; (ii) detecting the levelof expression or activity of the polypeptide in the pre-administrationsample; (iii) obtaining one or more post-administration samples from thesubject; (iv) detecting the level of expression or activity of thepolypeptide in the post-administration samples; (v) comparing the levelof expression or activity of the polypeptide in the pre-administrationsample with the polypeptide in the post-administration sample orsamples; and (vi) increasing or decreasing the administration of theagent to the subject accordingly.

Animal Models

B7-related polynucleotides such as BSL1 (e.g., SEQ ID NO:1 or 3), BSL2(e.g., SEQ ID NO:6, 10, 12, or 131), or BSL3 (e.g., SEQ ID NO:14) can beused to generate genetically altered non-human animals or human celllines. Any non-human animal can be used; however typical animals arerodents, such as mice, rats, or guinea pigs. Genetically engineeredanimals or cell lines can carry a gene that has been altered to containdeletions, substitutions, insertions, or modifications of thepolynucleotide sequence (e.g., exon sequence). Such alterations mayrender the gene nonfunctional, (i.e., a null mutation) producing a“knockout” animal or cell line. In addition, genetically engineeredanimals can carry one or more exogenous or non-naturally occurringgenes, e.g., “transgenes” or “orthologues”, that are derived fromdifferent organisms (e.g., humans), or produced by synthetic orrecombinant methods. Genetically altered animals or cell lines can beused to study BSL1, BSL2, or BSL3 function, regulation, and to developtreatments for BSL1-, BSL2-, or BSL3-related diseases. In particular,knockout animals and cell lines can be used to establish animal modelsand in vitro models for analysis of BSL1-, BSL2-, or BSL3-relateddiseases. In addition, transgenic animals expressing human BSL1, BSL2,or BSL3 can be used in drug discovery efforts.

A “transgenic animal” is any animal containing one or more cells bearinggenetic information altered or received, directly or indirectly, bydeliberate genetic manipulation at a subcellular level, such as bytargeted recombination or microinjection or infection with recombinantvirus. The term “transgenic animal” is not intended to encompassclassical cross-breeding or in vitro fertilization, but rather is meantto encompass animals in which one or more cells are altered by, orreceive, a recombinant DNA molecule. This recombinant DNA molecule maybe specifically targeted to a defined genetic locus, may be randomlyintegrated within a chromosome, or it may be extrachromosomallyreplicating DNA.

As used herein, the term “orthologue” denotes a gene or polypeptideobtained from one species that has homology to an analogous gene orpolypeptide from a different species. For example, the human BSL3 (e.g.,SEQ ID NO:15) and mouse AF142780 polypeptides are orthologues.

Transgenic animals can be selected after treatment of germline cells orzygotes. For example, expression of an exogenous BSL1, BSL2, or BSL3gene or a variant can be achieved by operably linking the gene to apromoter and optionally an enhancer, and then microinjecting theconstruct into a zygote (see, e.g., Hogan et al. (1994) Manipulating theMouse Embryo, A Laboratory Manual, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.). Such treatments include insertion of the exogenousgene and disrupted homologous genes. Alternatively, the gene(s) of theanimals may be disrupted by insertion or deletion mutation of othergenetic alterations using conventional techniques (see, e.g., Capecchi,(1989) Science, 244:1288; Valancuis et al. (1991) Mol. Cell Biol.,11:1402; Hasty et al. (1991) Nature, 350:243; Shinkai et al. (1992)Cell, 68:855; Mombaerts et al. (1992) Cell, 68:869; Philpott et al.(1992) Science, 256:1448; Snouwaert et al. (1992) Science, 257:1083;Donehower et al. (1992) Nature, 356:215).

In one aspect of the invention, BSL1, BSL2, or BSL3 knockout mice can beproduced in accordance with well-known methods (see, e.g., M. R.Capecchi, (1989) Science, 244:1288-1292; P. Li et al. (1995) Cell80:401-411; L. A. Galli-Taliadoros et al. (1995) J. Immunol. Methods181(1):1-15; C. H. Westphal et al. (1997) Curr. Biol. 7(7):530-3; S. S.Cheah et al. (2000) Methods Mol. Biol. 136:455-63). The human BSL1,BSL2, and BSL3 clones can be used isolate murine homologues. Murinehomologues can then be used to prepare a murine BSL1, BSL2, or BSL3targeting construct that can disrupt BSL1, BSL2, or BSL3 in the mouse byhomologous recombination at the corresponding chromosomal locus. Thetargeting construct can comprise a disrupted or deleted murine BSL1,BSL2, or BSL3 sequence that inserts in place of the functioning fragmentof the native mouse gene. For example, the construct can contain aninsertion in the murine BSL1, BSL2, or BSL3 protein-coding region.

Preferably, the targeting construct contains markers for both positiveand negative selection. The positive selection marker allows theselective elimination of cells that lack the marker, while the negativeselection marker allows the elimination of cells that carry the marker.In particular, the positive selectable marker can be an antibioticresistance gene, such as the neomycin resistance gene, which can beplaced within the coding sequence of murine BSL1, BSL2, or BSL3 torender it non-functional, while at the same time rendering the constructselectable. The herpes simplex virus thymidine kinase (HSV tk) gene isan example of a negative selectable marker that can be used as a secondmarker to eliminate cells that carry it. Cells with the HSV tk gene areselectively killed in the presence of gangcyclovir. As an example, apositive selection marker can be positioned on a targeting constructwithin the region of the construct that integrates at the BSL1, BSL2, orBSL3 locus. The negative selection marker can be positioned on thetargeting construct outside the region that integrates at the BSL1,BSL2, or BSL3 locus. Thus, if the entire construct is present in thecell, both positive and negative selection markers will be present. Ifthe construct has integrated into the genome, the positive selectionmarker will be present, but the negative selection marker will be lost.

The targeting construct can be employed, for example, in embryonal stemcell (ES). ES cells may be obtained from pre-implantation embryoscultured in vitro (M. J. Evans et al. (1981) Nature 292:154-156; M. O.Bradley et al. (1984) Nature 309:255-258; Gossler et al. (1986) Proc.Natl. Acad. Sci. USA 83:9065-9069; Robertson et al. (1986) Nature322:445-448; S. A. Wood et al. (1993) Proc. Natl. Acad. Sci. USA90:4582-4584). Targeting constructs can be efficiently introduced intothe ES cells by standard techniques such as DNA transfection or byretrovirus-mediated transduction. Following this, the transformed EScells can be combined with blastocysts from a non-human animal. Theintroduced ES cells colonize the embryo and contribute to the germ lineof the resulting chimeric animal (R. Jaenisch, (1988) Science240:1468-1474). The use of gene-targeted ES cells in the generation ofgene-targeted transgenic mice has been previously described (Thomas etal. (1987) Cell 51:503-512) and is reviewed elsewhere (Frohman et al.(1989) Cell 56:145-147; Capecchi (1989) Trends in Genet. 5:70-76;Baribault et al. (1989) Mol. Biol. Med. 6:481-492; Wagner, (1990) EMBOJ. 9:3025-3032; Bradley et al. (1992) Bio/Technology 10: 534-539).

Several methods can be used to select homologously recombined murine EScells. One method employs PCR to screen pools of transformant cells forhomologous insertion, followed by screening individual clones (Kim etal. (1988) Nucleic Acids Res. 16:8887-8903; Kim et al. (1991) Gene103:227-233). Another method employs a marker gene is constructed whichwill only be active if homologous insertion occurs, allowing theserecombinants to be selected directly (Sedivy et al. (1989) Proc. Natl.Acad. Sci. USA 86:227-231). For example, the positive-negative selection(PNS) method can be used as described above (see, e.g., Mansour et al.(1988) Nature 336:348-352; Capecchi (1989) Science 244:1288-1292;Capecchi, (1989) Trends in Genet. 5:70-76). In particular, the PNSmethod is useful for targeting genes that are expressed at low levels.

The absence of functional BSL1, BSL2, or BSL3 in the knockout mice canbe confirmed, for example, by RNA analysis, protein expression analysis,and functional studies. For RNA analysis, RNA samples are prepared fromdifferent organs of the knockout mice and the BSL1, BSL2, or BSL3transcript is detected in Northern blots using oligonucleotide probesspecific for the transcript. For protein expression detection,antibodies that are specific for the BSL1, BSL2, or BSL3 polypeptide areused, for example, in flow cytometric analysis, immunohistochemicalstaining, and activity assays. Alternatively, functional assays areperformed using preparations of different cell types collected from theknockout mice.

Several approaches can be used to produce transgenic mice. In oneapproach, a targeting vector is integrated into ES cell by homologousrecombination, an intrachromosomal recombination event is used toeliminate the selectable markers, and only the transgene is left behind(A. L. Joyner et al. (1989) Nature 338(6211):153-6; P. Hasty et al.(1991) Nature 350(6315):243-6; V. Valancius and O. Smithies, (1991) Mol.Cell Biol. 11(3):1402-8; S. Fiering et al. (1993) Proc. Natl. Acad. Sci.USA 90(18):8469-73). In an alternative approach, two or more strains arecreated; one strain contains the gene knocked-out by homologousrecombination, while one or more strains contain transgenes. Theknockout strain is crossed with the transgenic strain to produce newline of animals in which the original wild-type allele has been replaced(although not at the same site) with a transgene. Notably, knockout andtransgenic animals can be produced by commercial facilities (e.g., TheLerner Research Institute, Cleveland, Ohio; B&K Universal, Inc.,Fremont, Calif.; DNX Transgenic Sciences, Cranbury, N.J.; IncyteGenomics, Inc., St. Louis, Mo.).

Transgenic animals (e.g., mice) containing a nucleic acid molecule whichencodes human BSL1 (e.g., SEQ ID NO:2), BSL2 (e.g., SEQ ID NO:7, 11, or13), or BSL3 (e.g., SEQ ID NO:15) polypeptide, may be used as in vivomodels to study the effects of altered expression of BSL1, BSL2, orBSL3. Such animals can also be used in drug evaluation and discoveryefforts to find compounds effective to inhibit or modulate the activityof BSL1, BSL2, or BSL3, such as for example compounds for treatingimmune system disorders, diseases, or conditions. One having ordinaryskill in the art can use standard techniques to produce transgenicanimals which produce human BSL1, BSL2, or BSL3 polypeptide, and use theanimals in drug evaluation and discovery projects (see, e.g., U.S. Pat.No. 4,873,191 to Wagner; U.S. Pat. No. 4,736,866 to Leder).

In another embodiment of the present invention, the transgenic animalcan comprise a recombinant expression vector in which the nucleotidesequence that encodes human BSL1 (e.g., SEQ ID NO:2), BSL2 (e.g., SEQ IDNO:7, 11, or 13), or BSL3 (e.g., SEQ ID NO:15) is operably linked to atissue specific promoter whereby the coding sequence is only expressedin that specific tissue. For example, the tissue specific promoter canbe a mammary cell specific promoter and the recombinant protein soexpressed is recovered from the animal's milk.

In yet another embodiment of the present invention, a BSL1, BSL2, orBSL3 “knockout” can be produced by administering to the animalantibodies (e.g., neutralizing antibodies) that specifically recognizean endogenous BSL1, BSL2, or BSL3 polypeptide. The antibodies can act todisrupt function of the endogenous BSL1, BSL2, or BSL3 polypeptide, andthereby produce a null phenotype. In one specific example, a murineBSL1, BSL2, or BSL3 polypeptide or peptide can be used to generateantibodies. These antibodies can then be given to a mouse to knockoutthe function of the corresponding mouse protein.

In addition, non-mammalian organisms may be used to study BSL1, BSL2, orBSL3, and their related diseases. For example, model organisms such asC. elegans, D. melanogaster, and S. cerevisiae may be used. BSL1, BSL2,or BSL3 homologues can be identified in these model organisms, andmutated or deleted to produce a BSL1-, BSL2-, or BSL3-deficient strain.Human BSL1 (e.g., SEQ ID NO:2), BSL2 (e.g., SEQ ID NO:7, 11, or 13), orBSL3 (e.g., SEQ ID NO:15) can then be tested for the ability to“complement” the deficient strain. BSL1-, BSL2-, or BSL3-deficientstrains can also be used for drug screening. The study of BSL1, BSL2, orBSL3 homologues can facilitate the understanding of the biologicalfunction corresponding human gene, and assist in the identification ofbinding proteins (e.g., agonists and antagonists).

EMBODIMENTS

This invention encompasses, but is not limited to, the followingembodiments.

Section 1:

-   -   An isolated nucleic acid molecule encoding a polypeptide        comprising amino acid sequence SEQ ID NO:7.    -   An isolated nucleic acid molecule encoding a polypeptide        comprising amino acids 2-534 of SEQ ID NO:7.    -   An isolated nucleic acid molecule encoding a polypeptide        comprising amino acids 29-534 of SEQ ID NO:7.    -   An isolated nucleic acid molecule encoding a polypeptide        comprising at least 210 contiguous amino acids of SEQ ID NO:7.    -   An isolated nucleic acid molecule encoding a polypeptide        selected from the group consisting of a) a polypeptide        comprising amino acids 284-326 of SEQ ID NO:7; and b) a        polypeptide comprising amino acids 361-407 of SEQ ID NO:7.    -   An isolated nucleic acid molecule encoding a peptide selected        from the group consisting of a) a polypeptide comprising from        about amino acid 284 to about amino acid 290 of SEQ ID NO:7; b)        a peptide comprising from about amino acid 291 to about amino        acid 297 of SEQ ID NO:7; c) a peptide comprising from about        amino acid 298 to about amino acid 304 of SEQ ID NO:7; d) a        peptide comprising from about amino acid 305 to about amino acid        311 of SEQ ID NO:7; e) a peptide comprising from about amino        acid 312 to about amino acid 318 of SEQ ID NO:7; f) a peptide        comprising from about amino acid 319 to about amino acid 326 of        SEQ ID NO:7; g) a peptide comprising from about amino acid 361        to about amino acid 367 of SEQ ID NO:7; h) a peptide comprising        from about amino acid 368 to about amino acid 374 of SEQ ID        NO:7; i) a peptide comprising from about amino acid 375 to about        amino acid 381 of SEQ ID NO:7; j) a peptide comprising from        about amino acid 387 to about amino acid 393 of SEQ ID NO:7, k)        a peptide comprising from about amino acid 394 to about amino        acid 400 of SEQ ID NO:7; and 1) a peptide comprising from about        amino acid 401 to about amino acid 407 of SEQ ID NO:7.    -   An isolated nucleic acid molecule comprising a nucleotide        sequence selected from the group consisting of SEQ ID NO:6 and        131.    -   An isolated nucleic acid molecule comprising nucleotides        121-1722 of SEQ ID NO:6.    -   An isolated nucleic acid molecule comprising a nucleotide        sequence selected from the group consisting of a) a nucleotide        sequence comprising nucleotides 4-1602 of SEQ ID NO:131; and b)        a nucleotide sequence comprising nucleotides 124-1722 of SEQ ID        NO:6.    -   An isolated nucleic acid molecule comprising a nucleotide        sequence selected from the group consisting of a) a nucleotide        sequence comprising nucleotides 85-1602 of SEQ ID NO:131; and b)        a nucleotide sequence comprising nucleotides 205-1722 of SEQ ID        NO:6.    -   An isolated nucleic acid molecule comprising a nucleotide        sequence selected from the group consisting of a) a nucleotide        sequence comprising nucleotides 850-978 of SEQ ID NO:131; b) a        nucleotide sequence comprising nucleotides 1081-1221 of SEQ ID        NO:131; c) a nucleotide sequence comprising nucleotides 970-1098        of SEQ ID NO:6; and d) a nucleotide sequence comprising        nucleotides 1201-1341 of SEQ ID NO:6.    -   An isolated nucleic acid molecule comprising at least 630        contiguous nucleotides of SEQ ID NO:6.    -   An isolated nucleic acid molecule which is complementary to the        nucleic acid molecule of any one of the preceding embodiments in        section 1.    -   An isolated nucleic fusion acid molecule encoding amino acid        sequence SEQ ID NO:9.    -   An isolated nucleic acid fusion molecule comprising a) a        nucleotide sequence encoding amino acids 1-465 of SEQ ID NO:9;        and b) a nucleotide sequence encoding amino acids 466-698 of SEQ        ID NO:9.    -   An isolated nucleic acid fusion molecule comprising a) a        nucleotide sequence encoding amino acids 2-465 of SEQ ID NO:9;        and b) a nucleotide sequence encoding amino acids 466-698 of SEQ        ID NO:9.    -   An isolated nucleic acid fusion molecule comprising a) a        nucleotide sequence encoding amino acids 29-465 of SEQ ID NO:9;        and b) a nucleotide sequence encoding amino acids 466-698 of SEQ        ID NO:9.    -   An isolated nucleic acid fusion molecule comprising a) a        nucleotide sequence encoding at least 210 contiguous amino acids        of SEQ ID NO:7; and b) a nucleotide sequence encoding amino        acids 466-698 of SEQ ID NO:9.    -   An isolated nucleic acid fusion molecule comprising a)        nucleotide sequence comprising nucleotides 1-1394 of SEQ ID        NO:8; and b) a nucleotide sequence comprising nucleotides        1396-2094 of SEQ ID NO:8.    -   An isolated nucleic acid fusion molecule comprising a) a        nucleotide sequence comprising nucleotides 4-1394 of SEQ ID        NO:8; and b) a nucleotide sequence comprising nucleotides        1396-2094 of SEQ ID NO:8.    -   An isolated nucleic acid fusion molecule comprising a) a        nucleotide sequence comprising nucleotides 85-1394 of SEQ ID        NO:8; and b) a nucleotide sequence comprising nucleotides        1396-2094 of SEQ ID NO:8.    -   An isolated nucleic acid fusion molecule comprising a) a        nucleotide sequence comprising at least 630 contiguous        nucleotides of SEQ ID NO:6; and b) a nucleotide sequence        comprising nucleotides 1396-2094 of SEQ ID NO:8.    -   A vector comprising the nucleic acid molecule according to any        one of the preceding embodiments in section 1.    -   A vector comprising the nucleic acid fusion molecule according        to any one of the preceding embodiments in section 1.    -   A host cell comprising a vector that comprises the nucleic acid        molecule according to any one of the preceding embodiments in        section 1. In various embodiments, the host cell is selected        from the group consisting of bacterial, yeast, insect,        mammalian, and plant cells.    -   A host cell comprising a vector that comprises the nucleic acid        fusion molecule according to any one of the preceding        embodiments in section 1. In various embodiments, the host cell        is selected from the group consisting of bacterial, yeast,        insect, mammalian, and plant cells.    -   An isolated polypeptide comprising an amino acid sequence SEQ ID        NO:7.    -   An isolated polypeptide comprising amino acids 2-534 of SEQ ID        NO:7.    -   An isolated polypeptide comprising amino acids 29-534 of SEQ ID        NO:7.    -   An isolated polypeptide comprising at least 210 contiguous amino        acids of SEQ ID NO:7.    -   An isolated polypeptide selected from the group consisting of a)        a polypeptide comprising amino acids 284-326 of SEQ ID NO:7;        and b) a polypeptide comprising amino acids 361-407 of SEQ ID        NO:7.    -   An isolated peptide selected from the group consisting of a) a        peptide comprising from about amino acid 284 to about amino acid        290 of SEQ ID NO:7; b) a peptide comprising from about amino        acid 291 to about amino acid 297 of SEQ ID NO:7; c) a peptide        comprising from about amino acid 298 to about amino acid 304 of        SEQ ID NO:7; d) a peptide comprising from about amino acid 305        to about amino acid 311 of SEQ ID NO:7; e) a peptide comprising        from about amino acid 312 to about amino acid 318 of SEQ ID        NO:7; f) a peptide comprising from about amino acid 319 to about        amino acid 326 of SEQ ID NO:7; g) a peptide comprising from        about amino acid 361 to about amino acid 367 of SEQ ID NO:7; h)        a peptide comprising from about amino acid 368 to about amino        acid 374 of SEQ ID NO:7; i) a peptide comprising from about        amino acid 375 to about amino acid 381 of SEQ ID NO:7; j) a        peptide comprising from about amino acid 387 to about amino acid        393 of SEQ ID NO:7, k) a peptide comprising from about amino        acid 394 to about amino acid 400 of SEQ ID NO:7; and 1) a        peptide comprising from about amino acid 401 to about amino acid        407 of SEQ ID NO:7.    -   An isolated fusion polypeptide comprising amino acid sequence        SEQ ID NO:9.    -   An isolated fusion polypeptide comprising a) amino acids 1-465        of SEQ ID NO:9; and b) amino acids 466-698 of SEQ ID NO:9.    -   An isolated fusion polypeptide comprising a) amino acids 2-465        of SEQ ID NO:9; and b) amino acids 466-698 of SEQ ID NO:9.    -   An isolated fusion polypeptide comprising a) amino acids 29-465        of SEQ ID NO:9; and b) amino acids 466-698 of SEQ ID NO:9.    -   An isolated fusion polypeptide comprising a) at least 210        contiguous amino acids of SEQ ID NO:7; and b) amino acids        466-698 of SEQ ID NO:9.    -   An isolated antibody that binds to the polypeptide according to        the any one of the preceding embodiments in section 1. In a        specific aspect, the antibody is monoclonal.    -   An isolated antibody that binds to the peptide according to the        any one of the preceding embodiments in section 1. In a specific        aspect, the antibody is monoclonal.    -   An isolated antibody that binds to the fusion polypeptide        according to the any one of the preceding embodiments in        section 1. In a specific aspect, the antibody is monoclonal.    -   A hybridoma cell which produces the antibody according to any        one of the preceding embodiments in section 1.    -   A pharmaceutical composition comprising the nucleic acid        molecule according to any one of the preceding embodiments in        section 1, and a physiologically acceptable carrier, excipient,        or diluent.    -   A pharmaceutical composition comprising the nucleic acid fusion        molecule according to any one of the preceding embodiments in        section 1, and a physiologically acceptable carrier, excipient,        or diluent.    -   A pharmaceutical composition comprising the polypeptide        according to any one of the preceding embodiments in section 1,        and a physiologically acceptable carrier, excipient, or diluent.    -   A pharmaceutical composition comprising the peptide according to        any one of the preceding embodiments in section 1, and a        physiologically acceptable carrier, excipient, or diluent.    -   pharmaceutical composition comprising the fusion polypeptide        according to any one of the preceding embodiments in section 1,        and a physiologically acceptable carrier, excipient, or diluent.    -   A pharmaceutical composition comprising a host cell that        comprises the nucleic acid molecule according to any one of the        preceding embodiments in section 1, and a physiologically        acceptable carrier, excipient, or diluent.    -   A pharmaceutical composition comprising a host cell that        comprises the nucleic acid fusion molecule according to any one        of the preceding embodiments in section 1, and a physiologically        acceptable carrier, excipient, or diluent.    -   A pharmaceutical composition comprising an antibody that binds        to the polypeptide according to any one of the preceding        embodiments in section 1, and a physiologically acceptable        carrier, excipient, or diluent.    -   A pharmaceutical composition comprising an antibody that binds        to the peptide according to any one of the preceding embodiments        in section 1, and a physiologically acceptable carrier,        excipient, or diluent.    -   A pharmaceutical composition comprising an antibody that binds        to the fusion polypeptide according to any one of the preceding        embodiments in section 1, and a physiologically acceptable        carrier, excipient, or diluent.    -   A method of suppressing a T-cell-mediated immune response in a        subject comprising: administering to the subject at least one        pharmaceutical composition selected from the group consisting        of a) a pharmaceutical composition comprising the isolated        fusion polypeptide according to any one of the preceding        embodiments in section 1; b) a pharmaceutical composition        comprising the antibody according to any one or the preceding        embodiments in section 1; c) a pharmaceutical composition        comprising a host cell that comprises the nucleic acid fusion        molecule according to any one of the preceding embodiments in        section 1; and d) a pharmaceutical composition comprising the        nucleic acid fusion molecule according to any one of the        preceding embodiments in section 1; in an amount effective to        suppress the T-cell-mediated immune response.

In various aspects, the subject is affected with a condition selectedfrom the group consisting of tissue rejection, bone marrow rejection,organ transplant rejection, and graft versus host disease. In anotheraspect, the subject is affected with a condition associated withinflammation. In other aspects, the inflammation-associated condition isselected from the group consisting of psoriasis, chronic obstructivepulmonary disease, asthma, and atherosclerosis. In yet another aspect,the subject is affected with an autoimmune disease. In further aspects,the autoimmune disease is selected from the group consisting ofrheumatoid arthritis, multiple sclerosis, Lupus erythematosus,Hashimoto's thyroiditis, primary mixedema, Graves' disease, perniciousanemia, autoimmune atrophic gastritis, insulin dependent diabetesmellitus, good pasture's syndrome, myasthenia gravis, pemphigus, Crohn'sdisease, sympathetic opthalmia, autoimmune uveitis, autoimmune hemolyticanemis, idiopathic thrombocytopenia, primary biliary cirrhosis,ulcerative colitis, Sjogren's syndrome, polymyositis, and mixedconnective tissue disease.

-   -   A kit for detecting a B7-related nucleic acid molecule        comprising a) the isolated nucleic acid molecule according to        any one of the preceding embodiments in section 1; and b) at        least one component to detect binding of the isolated nucleic        acid molecule to the B7-related nucleic acid molecule.    -   A kit for detecting a B7-related nucleic acid molecule        comprising a) the isolated nucleic acid fusion molecule        according to any one of the preceding embodiments in section 1;        and b) at least one component to detect binding of the isolated        nucleic acid fusion molecule to the B7-related nucleic acid        molecule.    -   A kit for detecting a B7-related polypeptide comprising a) the        isolated antibody according to any one of the preceding        embodiments in section 1; and b) at least one component to        detect binding of the isolated antibody to a B7-related        polypeptide sequence.    -   A transgenic non-human animal comprising the nucleic acid        molecule according to any one of the preceding embodiments in        section 1.    -   A transgenic non-human animal comprising the nucleic acid fusion        molecule according to any one of the preceding embodiments in        section 1.    -   A cell line comprising the nucleic acid molecule according to        any one of the preceding embodiments in section 1.    -   A cell line comprising the nucleic acid fusion molecule        according to any one of the preceding embodiments in section 1.    -   An isolated BSL2vcvc nucleic acid molecule corresponding to ATCC        No. PTA-1993, deposited on Jun. 6, 2000.    -   An isolated BSL2vcvc-Ig nucleic acid fusion molecule        corresponding to ATCC Deposit No. ______, deposited Feb. 8,        2002.    -   An isolated BSL2vcvc polypeptide encoded by the nucleic acid        molecule corresponding to ATCC No. PTA-1993, deposited on Jun.        6, 2000.    -   An isolated BSL2vcvc-Ig fusion polypeptide encoded by the        nucleic acid fusion molecule corresponding to ATCC Deposit No.        ______, deposited Feb. 8, 2002.    -   A host cell comprising the BSL2vcvc nucleic acid molecule        corresponding to ATCC No. PTA-1993, deposited on Jun. 6, 2000.    -   A host cell comprising the BSL2vcvc-Ig nucleic acid fusion        molecule corresponding to ATCC Deposit No. ______, deposited        Feb. 8, 2002.    -   A hybridoma cell that produces antibodies to BSL2vcvc        corresponding to ATCC No. ______ deposited Feb. 8, 2002.    -   An anti-BSL2vcvc antibody produced by the hybridoma cell        corresponding to ATCC No. ______ deposited Feb. 8, 2002.

Section 2:

-   -   An isolated nucleic fusion acid molecule encoding amino acid        sequence SEQ ID NO:135.    -   An isolated nucleic acid fusion molecule comprising a) a        nucleotide sequence encoding amino acids 1-246 of SEQ ID NO:135;        and b) a nucleotide sequence encoding amino acids 249-480 of SEQ        ID NO:135.    -   An isolated nucleic acid fusion molecule comprising a) a        nucleotide sequence encoding amino acids 2-246 of SEQ ID NO:135;        and b) a nucleotide sequence encoding amino acids 249-480 of SEQ        ID NO:135.    -   An isolated nucleic acid fusion molecule comprising a) a        nucleotide sequence encoding amino acids 29-246 of SEQ ID        NO:135; and b) a nucleotide sequence encoding amino acids        249-480 of SEQ ID NO:135.    -   An isolated nucleic acid fusion molecule comprising a) a        nucleotide sequence encoding at least 90 contiguous amino acids        of SEQ ID NO:11; and b) a nucleotide sequence encoding amino        acids 249-480 of SEQ ID NO:135.    -   An isolated nucleic acid fusion molecule comprising a)        nucleotide sequence comprising nucleotides 1-738 of SEQ ID        NO:134; and b) a nucleotide sequence comprising nucleotides        745-1440 of SEQ ID NO:134.    -   An isolated nucleic acid fusion molecule comprising a) a        nucleotide sequence comprising nucleotides 4-738 of SEQ ID        NO:134; and b) a nucleotide sequence comprising nucleotides        745-1440 of SEQ ID NO:134.    -   An isolated nucleic acid fusion molecule comprising a) a        nucleotide sequence comprising nucleotides 85-738 of SEQ ID        NO:134; and b) a nucleotide sequence comprising nucleotides        745-1440 of SEQ ID NO:134.    -   An isolated nucleic acid fusion molecule comprising a) a        nucleotide sequence comprising at least 270 contiguous        nucleotides of SEQ ID NO:10; and b) a nucleotide sequence        comprising nucleotides 745-1440 of SEQ ID NO:134.    -   A vector comprising the nucleic acid fusion molecule according        to any one of the preceding embodiments in section 2.    -   A host cell comprising a vector that comprises the nucleic acid        fusion molecule according to any one of the preceding        embodiments in section 2. In various embodiments, the host cell        is selected from the group consisting of bacterial, yeast,        insect, mammalian, and plant cells.    -   An isolated fusion polypeptide comprising amino acid sequence        SEQ ID NO:135.    -   An isolated fusion polypeptide comprising a) amino acids 1-246        of SEQ ID NO:135; and b) amino acids 249-480 of SEQ ID NO:135.    -   An isolated fusion polypeptide comprising a) amino acids 2-246        of SEQ ID NO:135; and b) amino acids 249-480 of SEQ ID NO:135.    -   An isolated fusion polypeptide comprising a) amino acids 29-246        of SEQ ID NO:135; and b) amino acids 249-480 of SEQ ID NO:135.    -   An isolated fusion polypeptide comprising a) at least 90        contiguous amino acids of SEQ ID NO:11; and b) amino acids        249-480 of SEQ ID NO:135.    -   An isolated antibody that binds to the fusion polypeptide        according to the any one of the preceding embodiments in        section 2. In a specific aspect, the antibody is monoclonal.    -   A hybridoma cell which produces the antibody according to any        one of the preceding embodiments in section 2.    -   A pharmaceutical composition comprising the nucleic acid fusion        molecule according to any one of the preceding embodiments in        section 2, and a physiologically acceptable carrier, excipient,        or diluent.    -   A pharmaceutical composition comprising the fusion polypeptide        according to any one of the preceding embodiments in section 2,        and a physiologically acceptable carrier, excipient, or diluent.    -   A pharmaceutical composition comprising a host cell according to        any one of the preceding embodiments in section 2, and a        physiologically acceptable carrier, excipient, or diluent.    -   A pharmaceutical composition comprising the antibody according        to any one of the preceding embodiments in section 2, and a        physiologically acceptable carrier, excipient, or diluent.    -   An isolated BSL2v2c2 nucleic acid fusion molecule corresponding        to ATCC Deposit No. ______, deposited Feb. 8, 2002.    -   An isolated BSL2v2c2 fusion polypeptide encoded by the nucleic        acid fusion molecule corresponding to ATCC Deposit No. ______,        deposited Feb. 8, 2002.    -   A host cell comprising the BSL2v2c2 nucleic acid fusion molecule        corresponding to ATCC Deposit No. ______, deposited Feb. 8,        2002.

Section 3:

-   -   An isolated nucleic fusion acid molecule encoding amino acid        sequence SEQ ID NO:133.    -   An isolated nucleic acid fusion molecule comprising a) a        nucleotide sequence encoding amino acids 1-226 of SEQ ID NO:133;        and b) a nucleotide sequence encoding amino acids 229-480 of SEQ        ID NO:133.    -   An isolated nucleic acid fusion molecule comprising a) a        nucleotide sequence encoding amino acids 2-226 of SEQ ID NO:133;        and b) a nucleotide sequence encoding amino acids 229-480 of SEQ        ID NO:133.    -   An isolated nucleic acid fusion molecule comprising a) a        nucleotide sequence encoding amino acids 29-226 of SEQ ID NO:13;        and b) a nucleotide sequence encoding amino acids 229-480 of SEQ        ID NO:133.    -   An isolated nucleic acid fusion molecule comprising a) a        nucleotide sequence encoding at least 170 contiguous amino acids        of SEQ ID NO:13; and b) a nucleotide sequence encoding amino        acids 229-480 of SEQ ID NO:133.    -   An isolated nucleic acid fusion molecule comprising a)        nucleotide sequence comprising nucleotides 1-738 of SEQ ID        NO:132; and b) a nucleotide sequence comprising nucleotides        745-1440 of SEQ ID NO:132.    -   An isolated nucleic acid fusion molecule comprising a) a        nucleotide sequence comprising nucleotides 4-738 of SEQ ID        NO:132; and b) a nucleotide sequence comprising nucleotides        745-1440 of SEQ ID NO:132.    -   An isolated nucleic acid fusion molecule comprising a) a        nucleotide sequence comprising nucleotides 85-738 of SEQ ID        NO:132; and b) a nucleotide sequence comprising nucleotides        745-1440 of SEQ ID NO:132.    -   An isolated nucleic acid fusion molecule comprising a) a        nucleotide sequence comprising at least 410 contiguous        nucleotides of SEQ ID NO:12; and b) a nucleotide sequence        comprising nucleotides 745-1440 of SEQ ID NO:132.    -   A vector comprising the nucleic acid fusion molecule according        to any one of the preceding embodiments in section 3.    -   A host cell comprising a vector that comprises the nucleic acid        fusion molecule according to any one of the preceding        embodiments in section 3. In various embodiments, the host cell        is selected from the group consisting of bacterial, yeast,        insect, mammalian, and plant cells.    -   An isolated fusion polypeptide comprising amino acid sequence        SEQ ID NO:133.    -   An isolated fusion polypeptide comprising a) amino acids 1-226        of SEQ ID NO:133; and b) amino acids 229-480 of SEQ ID NO:133.    -   An isolated fusion polypeptide comprising a) amino acids 2-226        of SEQ ID NO:133; and b) amino acids 229-480 of SEQ ID NO:133.    -   An isolated fusion polypeptide comprising a) amino acids 29-226        of SEQ ID NO:133; and b) amino acids 229-480 of SEQ ID NO:133.    -   An isolated fusion polypeptide comprising a) at least 170        contiguous amino acids of SEQ ID NO:13; and b) amino acids        229-480 of SEQ ID NO:133.    -   An isolated antibody that binds to the fusion polypeptide        according to the any one of the preceding embodiments in        section 3. In a specific aspect, the antibody is monoclonal.    -   A hybridoma cell which produces the antibody according to any        one of the preceding embodiments in section 3.    -   A pharmaceutical composition comprising the nucleic acid fusion        molecule according to any one of the preceding embodiments in        section 3, and a physiologically acceptable carrier, excipient,        or diluent.    -   A pharmaceutical composition comprising the fusion polypeptide        according to any one of the preceding embodiments in section 3,        and a physiologically acceptable carrier, excipient, or diluent.    -   A pharmaceutical composition comprising a host cell according to        any one of the preceding embodiments in section 3, and a        physiologically acceptable carrier, excipient, or diluent.    -   A pharmaceutical composition comprising the antibody according        to any one of the preceding embodiments in section 3, and a        physiologically acceptable carrier, excipient, or diluent.    -   An isolated BSL2v1c2 nucleic acid fusion molecule corresponding        to ATCC Deposit No. ______, deposited Feb. 8, 2002.    -   An isolated BSL2v1c2 fusion polypeptide encoded by the nucleic        acid fusion molecule corresponding to ATCC Deposit No. ______,        deposited Feb. 8, 2002.    -   A host cell comprising the BSL2v1c2 nucleic acid fusion molecule        corresponding to ATCC Deposit No. ______, deposited Feb. 8,        2002.

EXAMPLES

The examples as set forth herein are meant to exemplify the variousaspects of the present invention and are not intended to limit theinvention in any way.

Example 1 Identification of BSL1

Preparation of monocytes: Human monocytes were obtained from peripheralblood mononuclear cells by elutriation. The elutriation buffer contained1 L RPMI (GibcoBRL/Life Technologies Inc., Rockville, Md.; Cat.#11875-085) with 2.5 mM EDTA and 10 μg/ml polymyxin B. The buffer wasprepared 1 day prior to the elutriation procedure, and stored at roomtemperature. For the elutriation procedure, 225 ml EDTA wholeblood/donor was obtained and prepared. Twenty milliliters of elutriationbuffer was added to twelve 50 ml centrifuge tubes, and the 225 ml ofblood was divided equally among the tubes. Twelve milliliters of ficollsolution (lymphocyte separation medium) was used to underlay the mixturein each tube (AccuPrep Lymphocytes, Accurate Scientific Co.; Cat. #1053980). The tubes were centrifuged at 1800 rpm for 25 min (minutes).

Sheep's red blood cells (SRBC) were prepared by the following procedure.Twelve milliliters of Sheep Blood Alsever's (Colorado Serum Co., Denver,Colo.; Cat. # CS1112) was resuspended, and centrifuged at 2000 rpm for10 min. The top coating of the SRBC pellet was removed, and the pelletwas washed 2 times with elutriation buffer. Following each wash, thepellet was centrifuged at 2200 rpm for 8 min. After the second wash, thepellet was resuspended in 5 ml elutriation buffer and refrigerated.

Following centrifugation, the supernatant from the ficoll underlay tubeswas aspirated to leave behind approximately 5 ml of the interface layer.The interface layer was then carefully removed to a new tube, withoutdisturbing the red cell pellet in each tube. The interface layers of two50 ml tubes was combined and transferred to a new 50 ml tube, resultingin a total of six 50 ml tubes. Elutriation buffer was added to each tubeto bring the final volume to 50 ml per tube. The tubes were centrifugedat 1800 rpm for 10 min, and the supernatant was removed. The peripheralblood monocytes from each donor were combined and divided between twotubes, and resuspended in 40 ml elutriation buffer per tube. The tubeswere centrifuged at 1500 rpm for 10 min, the supernatant was aspirated,and the pellet was resuspended in 30 ml elutriation buffer per tube.

Following this step, 3 ml of washed SRBC was added to each tube, and themixture was centrifuged at 1000 rpm for 5 min. The pellet was incubatedon ice for at least 1 hr (hour), and each pellet was gently resuspendedby inverting the tubes. Twelve milliliters of ficoll was used tounderlay the mixture in each tube. The tubes were centrifuged at 2500rpm for 20 min, and the interface layers were removed to new tubes aspreviously described. The interface layers were then resuspended in 10ml elutriation buffer and stored on ice until the start of theelutriation procedure. It is noted the purification of T-cells usingtheir affinity to SRBCs is termed E-rosetting.

Elutriation of monocytes: Elutriation was performed as follows. Theelutriation pump speed was set to ˜65 ml/min, and the storage ethanolwas removed from the feed lines for the pump. The feed lines, chambers,and rotor were washed with ˜100 ml distilled water. Pump speed wasreduced to 50 ml/min, and elutriation buffer was passed through the feedlines, chamber, and rotor. The feed lines were checked for the presenceof bubbles, and any bubbles were removed. The centrifuge speed was thenset at 1950±1 rpm, and the pump was calibrated at 15 ml/min. The pumpspeed was then reduced to 11 ml/min and the stopcock to the chamber wasclosed.

To load cells, the loading syringe stopcock was closed and the outletpipette was placed in the 50 ml sample tube labeled as 11 ml/minfraction. The cells were mixed and added to the loading syringe. Thestopcock of the loading syringe was opened and the feeding tube wasrinsed with 10 ml elutriation buffer. When ˜0.5 ml cells remained in thesyringe, elutriation buffer was added and this step was repeated onemore time. Following this step, the loading syringe stopcock was closedand the chamber stopcock was opened.

Fifty milliliter fractions were collected at 11 (2 fractions), 14, 16,and 36 (2 fractions) ml/min pump speeds, and then placed on ice. Inaccordance with previous observations, the monocytes were predicted tobe in the 36 ml/min fraction. The monocyte fraction was centrifuged at1800 rpm for 8 min, and the cells were resuspended in 10 ml 25% fetalbovine serum/RPMI with 1 μg/ml polymyxin B. The cells were counted andstored on ice until use.

Cell culture conditions: The isolated peripheral blood monocytes wereresuspended in RPMI 1640 medium containing 10% fetal bovine serum(Hyclone, Logan, Utah) supplemented with penicillin/streptomycin, 2 mMglutamine, 1% non-essential amino acids, 1 mM sodium pyruvate, and IL-4(75 ng/ml) and GM-CSF (15 ng/ml) cytokines (each from GibcoBRL). Thecell suspension (5×10⁵ cells/ml) was transferred to tissue cultureflasks and incubated in chambers containing 5% CO₂ at 37° C. for 7 days.Following the incubation period, the cell cultures were pipettedvigorously to remove cells from the flask. The human monocytic cell lineTHP1 was grown at 37° C. in 5% CO₂ to a final concentration of 5×10⁵cells/ml in RPMI 1640 medium containing 10% fetal bovine serumsupplemented with penicillin/streptomycin and 2 mM glutamine.

Poly(A)⁺ RNA isolation: GM-CSF/IL-4 differentiated human peripheralblood mononuclear cells (2×10⁸ cells) and THP1 human monocytes (2×10⁸cells) were washed twice with PBS (GibcoBRL) at 4° C. Poly(A)⁺ RNA wasisolated directly using the Fast Track™ 2.0 kit (Invitrogen Corp.,Carlsbad, Calif.).

Subtraction library construction: A cDNA subtraction library was madeusing the CLONTECH PCR-Select™ cDNA Subtraction Kit (CLONTECH, PaloAlto, Calif.). Manufacturer's protocols were followed using 2.0 μg ofGM-CSF/IL-4 differentiated human peripheral blood monocyte poly (A)⁺ RNAas the tester sample and 2.0 μg of resting THP1 monocyte poly(A)⁺ RNA asthe driver sample. Ten secondary PCRs were combined and run on a 1.2%agarose gel. Fragments ranging from approximately 0.3-kb to 1.5-kb weregel purified using the QIAGEN gel extraction kit (QIAGEN, Valencia,Calif.) and inserted into the TA cloning vector, pCR2.1 (Invitrogen).The construct was used for transformation into TOP10F′ competent E. coli(Invitrogen), and transformants were plated onto Lauria-Bertani (LB)plates containing 50 μg/ml ampicillin. Approximately 300 clones wereisolated and grown in LB broth containing similar concentrations ofampicillin. Plasmids were isolated using QIAGEN miniprep spin (QIAGEN)and sequenced using ABI cycle sequencers (ABI Prism, PE AppliedBiosystems).

Full-length cloning: To clone the 5′ and 3′ ends of BSL1, the SMART™RACE (rapid amplification of cDNA ends) cDNA Amplification kit(CLONTECH) was used according to the manufacturer's directions. The 5′and 3′ RACE libraries were constructed using 1.0 μg of poly(A)⁺ RNAtemplate obtained from human microvascular endothelial cell treated withTNF-alpha for 1 hr. The 5′-RACE-PCR mixture contained 1.5 μl of 5′-RACEready cDNA, 0.4 μM JNF 155 primer (5′-GGCATAATAAGATGGCTCCC-3′; SEQ IDNO:21), 1× Universal Primer Mix (UPM), 200 μM dNTP, 1× Advantaq Plus PCRbuffer (CLONTECH), and 1× Advantaq Plus Polymerase (CLONTECH) in a totalvolume of 25 μl. The 3′-RACE-PCR mixture contained the same bufferconditions, 3′-RACE ready cDNA, and 0.4 μM JNF 154 primer(5′-CATGAACTGACATGTCAGGC-3′; SEQ ID NO:22). Both reactions wereincubated using a traditional touchdown PCR approach: 5 cycles ofincubation at 94° C. for 30 sec (seconds), 65° C. for 30 sec, and 72° C.for 3 min; 5 cycles of incubation at 94° C. for 30 sec, 63° C. for 30sec, and 72° C. for 3 min; and 15 cycles of incubation at 94° C. for 30sec, 62° C. for 30 sec, and 72° C. for 3 min.

The PCR products were isolated by electrophoresis using a 2.0% agarosegel, and DNA was visualized by ethidium bromide staining. An 888-bpfragment from the 5′-RACE reaction and a 1,110-bp fragment from the3′-RACE reaction were purified using the QIAGEN gel extraction kit andresuspended in 10 μl distilled water. Six microliters of each fragmentwas ligated into pCR2.1-TA cloning vector (Invitrogen), and the ligationmixture was used for transformation into TOP10F′ ultracompetent E. colicells (Invitrogen). Transformants were plated onto LB platessupplemented with 50 μg/ml ampicillin, 40 mg/ml X-gal, and 100 mM IPTG.Colonies were isolated and grown overnight at 37° C. in 4 ml of LB-brothsupplemented with 50 μg/ml ampicillin. Plasmids were isolated using theQIAGEN miniprep spin kit (QIAGEN), resuspended in 30 μl distilled water,and sequenced using an ABI cycle sequencer (ABI Prism, PE AppliedBiosystems).

To generate the full-length clone, JNF155RACE5.1, JNF154RACE3.2, andpCR2.1 were ligated together. JNF155RACE5.1 was doubly digested withXhoI and HindIII. JNF154RACE3.2 was doubly digested with HindIII andEcoRI. The TA cloning vector (Invitrogen) was digested with XhoI andEcoRI. Each fragment was purified using the QIAGEN gel extraction kitand resuspended in water. One microliter of each digested fragment wasligated together in the same reaction using T4 DNA ligase. The ligationmixture was used for transformation into TOP10F′ E. coli ultracompetentcells. Transformants were plated onto LB plates containing 50 μg/mlampicillin, 40 mg/ml X-gal, and 100 mM IPTG. Colonies were isolated fromthe plates, and grown in LB broth containing 50 μg/ml ampicillinovernight at 37° C. Plasmids containing full-length BSL1 were purifiedusing the QIAGEN miniprep spin kit (QIAGEN), and sequenced (ABI cyclesequencer; PE Applied Biosystems). The plasmid carrying DNA encoding thefull-length BSL1 sequence (pTADV:BSL1) was deposited with the AmericanType Culture Collection (ATCC, 10801 University Blvd., Manassas, Va.20110-2209 USA), under ATCC Designation No. PTA-1989, on Jun. 6, 2000.

Example 2 Characterization of BSL1

Sequence analysis of the BSL1 clones: The full-length BSL1 nucleotideand predicted amino acid sequence was determined from a clone isolatedfrom TNF-alpha treated human microvascular endothelial cell cDNAsubtraction library (FIGS. 1A-1B) and a clone isolated from aGM-CSF/IL-4 differentiated human monocyte cDNA library (FIG. 1C). Thesequencing primers for the BSL1 clones are shown in Table 1. TABLE 1Primer Sequence SEQ ID NO: JNF 292 forward CATTTACAAAGAGAGGTCGG 23 JNF298 reverse AGGGTTATTTTAAGTACCGACC 24 JNF 293 forwardGGAAATGTATGTTAAAAGCACG 25 JNF 297 reverse GGCATGGATCCTCAGCCCTGGG 26 JNF294 forward GAGACCCATGGGCTCTCCAGGG 27 JNF 296 reverseGTTCAAGCACAACGAATGAGGC 28 JNF 295 forward TGGCTTTGCCACATGTCAAGGC 29

Both BSL1 clones had identical coding sequences, however, the cloneobtained from the differentiated human monocyte cDNA library contained adifferent sequence in the 3′ untranslated region of the BSL1 gene (FIG.2B; see bold text). The nucleotide and predicted amino acid sequences ofBSL1 are shown in FIGS. 1A-1C.

EST clones encoding BSL1 were identified from public (GenBank) andprivate (Incyte Genomics) databases, and are shown in Table 2. TABLE 2Length Position Clone ID Database Tissue (bp) (1-3797) AI733919 GenBankOvary tumor 429 401-829 AA292201 GenBank Ovary tumor 430 401-830AA399416 GenBank Ovary tumor 325 506-830 3166966H1 Incyte CD4⁺ T lymphost/CD3, CD28 Ab's 197 415-611 4415633H1 Incyte Peripheral BloodMonocytes, t/anti- 253 542-794 IL-10, LPS AA368815 GenBank Placenta,fetal 55  998-1052 5611256H1 Incyte Peripheral Blood Monocytes, t/anti-254 1005-1258 IL-10, LPS, SUB 5048659F6 Incyte Placenta, fetal 3321016-1347 3680369H1 Incyte Lung, aw/asthma 240 1203-1442 AI202916GenBank Germ cell tumor, pool, SUB 259 1381-1639 AA373164 GenBank Lungfibroblast line, HSC172, fetal 274 1416-1689 4354914H1 Incyte Fat,auxiliary, aw/breast adenoCA 288 1449-1736 AA037078 GenBank Fibroblastssenescent 365 1529-1893 171033R6 Incyte Bone Marrow 273 1785-2057R30906/ GenBank Placenta, neonatal 1932 1867-3798 R30861**Full-length sequence not known.

It is noted that the BSL1 coding sequence and predicted amino acidsequence have also been identified as B7-H1 and PD-L1 (H. Dong et al.(1999) Nature Med. 5:1365-9; GenPept Accession No. NP_(—)054862; G. J.Freeman et al. (2000) J. Exp. Med. 192:1027-1034). In addition, a murinehomologue of B7-H1 has been identified (H. Tamura et al. (2001) Blood97:1809-1816). Notably, the mouse and human B7-H1 factors have beendescribed as costimulatory molecules (H. Dong et al. (1999) Nature Med.5:1365-9; H. Tamura et al. (2001) Blood 97:1809-1816), whereas PD-L1 hasbeen described as an inhibitor of T-cell proliferation (G. J. Freeman etal. (2000) J. Exp. Med. 192:1027-1034).

Chromosomal Mapping: BSL1 was previously mapped utilizing radiationhybrid mapping (T. Ishida et al. (1999) CytoGenet. Cell Genet.85:232-6). Analysis of NCBI's Genemap '99 using GenBank EST AA399416indicated that the BSL1 gene was linked to chromosome 9p24 with theorder of AFM274xe1-stSG46389 (BSL1)-AFM242xh6.

BSL1 expression analysis: BSL1 expression patterns were determined byNorthern blot analysis of several cell types, including restingperipheral blood T-cells, peripheral blood T-cells stimulated withanti-CD3/anti-CD28 antibodies, peripheral blood T-cells stimulated withphorbol 12 myristate 13 acetate (PMA), peripheral blood T-cellsstimulated with phytohemaglutinin (PHA), resting THP1 monocytes, THP1stimulated with lipopolysacharide (LPS), resting peripheral bloodmonocytes, resting peripheral blood monocytes stimulated with PHA,resting peripheral blood monocytes stimulated with GM-CSF and IL-4, RAJIB cells, RAMOS B cells, resting human microvascular endothelial cells(HMVEC), HMVEC stimulated with TNF-alpha, and serum starved H292 humanlung epithelial cells.

Cell culture conditions: Peripheral blood T-cells were grown in RPMI1640 (Hyclone) with 10% human serum at 37° C. in 5% CO₂ for 48 hr.Peripheral blood T-cells stimulated with anti-CD3 and anti-CD28antibodies were grown in RPMI 1640 with 10% human serum at 37° C. in 5%CO₂ for 24-72 hr in the presence of 1 μg/ml anti-CD3 monoclonalantibodies (MAb G19.4; P. S. Linsley et al. (1993) Ann. Rev. Immunol.11:191-212) and 1 μg/ml anti-CD28 monoclonal antibodies (MAb 9.3;Linsley et al., supra). Peripheral blood T-cells stimulated with PMA andionomycin were grown in RPMI 1640 (Hyclone) with 10% human serum at 37°C. in 5% CO₂ for 48 hr in the presence of 30 ng/ml PMA with 1 μMionomycin. Peripheral blood T-cells stimulated with PHA were grown inRPMI 1640 (Hyclone) with 10% human serum at 37° C. in 5% CO₂ for 48 hrin the presence of 3 μg/ml PHA. THP1 cells obtained from an immortalhuman monocytic cell line were grown in RPMI 1640 (Hyclone) with 10%fetal bovine serum at 37° C. in 5% CO₂ with or without 100 ng/ml LPS for2 hr. Peripheral blood monocytes were grown in RPMI 1640 (Hyclone) with25% fetal bovine serum in teflon plates at 37° C. in 5% CO₂ with orwithout 1 μg/ml PHA or 15 ng/ml GM-CSF with 75 ng/ml IL-4 for 7 days.RAJI and RAMOS cells obtained from immortal human B cell lines weregrown in RPMI 1640 (Hyclone) with 10% fetal bovine serum at 37° C. in 5%CO₂. HMVEC were grown in DMEM with 10% fetal bovine serum at 37° C. in5% CO₂ with or without 10 ng/ml TNF-alpha for 1-24 hr. H292 cellsobtained from an immortal human lung epithelial cell line were grown inRPM1 1640 (Hyclone) with 10% fetal bovine serum at 37° C. in 5% CO₂, andthen grown in serum free medium for 16 hr prior to harvest.

Northern blot analysis: For Northern blot analysis, 0.5 μg of totalpoly(A)⁺ RNA obtained from each cell type was separated on a 1.2%agarose gel containing 3% formaldehyde, and transferred to a Hybond-N+nylon membrane (Amersham) overnight using 20×SSC as transfer buffer. Themembrane was then auto-crosslinked, washed with 4×SSPE, and allowed toair-dry. The membrane was then prehybridized at 65° C. in ExpressHybsolution (CLONTECH) for 1 hr, and then hybridized with a[³²P]-dCTP-radiolabeled (NEN, Boston, Mass.) random primed BSL1 cDNAprobe. The probe was obtained from a 666-bp BSL1 HindIII/PstI fragment(FIG. 6A), which was purified using the NucTrap purification column(Stratagene), and radiolabeled to have a specific activity of 2.0×10⁶cpm/ml. Following hybridization, the membrane was washed in 2.0×SSC with0.05% SDS at 65° C., and exposed to film for 72 hr at −70° C.

A 3.8-kb BSL1 mRNA transcript was detected in several cell types. Inparticular, high levels of BSL1 mRNA were detected in peripheral bloodmonocytes stimulated with PHA, and in HMVEC stimulated with TNF-alpha(FIG. 7D). Moderate levels of BSL1 mRNA were detected in peripheralblood T-cells following stimulation with anti-CD3 and anti-CD28monoclonal antibodies for 72 hr (FIG. 7D). Moderate levels of BSL1 mRNAwere also observed in THP1 cells stimulated with LPS (FIG. 7D). However,BSL1 mRNA was not detected in resting THP1 cells, resting BJAB cells,LPS-activated BJAB cells, resting peripheral blood T-cells,PBT-activated peripheral blood T-cells, or GM-CSF/IL-4-activatedperipheral blood monocytes (FIG. 7D). In addition, BSL1 mRNA was notdetected in resting RAJI cells, resting RAMOS cells, or serum starvedH292 cells (FIG. 7D).

BSL1-Ig fusion construct: The DNA fragment corresponding to the BSL1predicted extracellular domain (ECD; amino acids 23-290 of SEQ ID NO:12)was amplified by PCR utilizing full-length BSL1-pCR2.1 as a template,and oligonucleotide primers that hybridized to the 5′ and 3′ ends of theBSL1 ECD: JNF 184 forward primer5′-TCAGGTACTAGTGTTCCCAAGGACCTATATGTGG-3′; SEQ ID NO:30); and JNF 185reverse primer (5′-GATTCGAGATCTCCTCGAGTCCTTTCATTTGGAGGATGTGCC-3′ (SEQ IDNO:31). PCR was performed using ˜100 ng template DNA, 0.4 μM of eachprimer, 200 μM dNTP, 1× Advantage 2 PCR buffer, and 1× Advantage 2Polymerase (CLONTECH) in a total volume of 50 μl. The PCR mixture wasincubated at 94° C. for 30 sec, 62° C. for 30 sec, and 72° C. for 1 min,and this was repeated for 30 cycles. The PCR products were separated bygel electrophoresis on a 1.2% agarose gel, and the DNA was visualized byethidium bromide staining. A 680-bp fragment corresponding to the BSL1ECD was purified from the agarose gel using the QIAGEN gel extractionkit (QIAGEN), and resuspended in 32 μl distilled water.

The BSL1 ECD fragment was then digested using SpeI and BglII restrictionendonucleases and directionally cloned into SpeI/BamHI-digested PD19vector using 5 units (U)/μl T4 DNA ligase (GibcoBRL). The ligationmixture was used for transformation into DH5-alpha competent E. colicells (GibcoBRL), and transformants were plated onto Lauria-Bertani (LB)plates containing 50 μg/ml ampicillin. Plates were incubated overnightat 37° C., and colonies were isolated and grown overnight at 37° C. inLB broth containing 50 μg/ml ampicillin. Plasmids were isolated usingthe QIAGEN miniprep spin kit, resuspended in 50 μl distilled water, andsequenced using ABI cycle sequencer (PE Biosystems, Foster City,Calif.). Primer sequences were as follows: sense JNF 184(5′-TCAGGTACTAGTGTTCCCAAGGACCATATGTGG-3′; SEQ ID NO:32) and anti-senseJNF 185 (5′-GATTCGAGATCTCCTCGAGTCTTTCATTGGGGATGTGCC-3′; SEQ ID NO:33).

The plasmid carrying DNA encoding BSL1-Ig (pD19:BSL1Ig) was depositedwith the American Type Culture Collection (ATCC, 10801 University Blvd.,Manassas, Va. 20110-2209 USA), under ATCC Designation No. PTA-1992, onJun. 6, 2000. The nucleotide and predicted amino acid sequence ofBSL1-Ig is shown in FIGS. 2A-2B.

BSL1 monoclonal antibodies: The BSL1-Ig fusion protein was purified byaffinity purification as described for BSL3-Ig, below. The purifiedfusion protein was then used to immunize mice using the protocoldescribed for BSL3-Ig. Following this, hybridoma cell lines wereconstructed and BSL1 monoclonal antibodies (MAbs) were isolated asdescribed for BSL3, below. In addition, BSL1 MAbs were screened forspecificity by whole-cell ELISA. For whole cell ELISA, COS cells weretransiently transfected with full length BSL1 (L156-3). Cells werelifted with versene on day 4 following transfection. Cells were washedtwice in PBS and resuspended in PBS with 10% FBS at a concentration of5.0×10⁶ cells/ml. Then, 50 μl of cells were added to each well of aFalcon 3911 96-well plate and incubated on ice for 30 min. Next, 50 μlsupernatant from the putative BSL1 hybridomas was added per well andincubated on ice for 30 min. Cells were washed twice in PBS. Cells werethen resuspended in goat anti-mouse HRP-conjugated secondary antibodies(Amersham; Cat. # NA9310) diluted 1:1000 in PBS. Cells were incubated onice for 30 min, washed twice in PBS, and resuspended in 125 μl PBS.Following this, cells were transferred to a fresh plate. Cells werewashed in PBS and resuspended in 25 μl PBS. Next, 125 μl Peroxidasesolution B (KPL, Gaithersburg, Md.; Cat. #50-65-00) with TMB peroxidasesubstrate (KPL; Cat. # 50-76-01) was added. Color was allowed todevelop. Cells were pelleted, and 100 μl supernatant was transferred toan Immulon 2 plate. The signal was quenched with 100 μl 1 N sulfuricacid, and the plates were read at OD₄₅₀/OD₆₃₀.

Example 3 Identification of BSL2

Database searches: BSL2 was identified by BLAST and FASTA analysis ofthe Incyte Genomics sequence databases (Incyte Genomics) utilizing theB7-1 or B7-2 amino acid sequences as query sequences. For BLASTanalysis, the BLOSSUM-62 scoring matrix was used (S. Henikoff et al.(1992) Proc. Natl. Acad. Sci. USA 89:10915-10919), and the remainingparameters were set to the default designations. For FASTA analysis, allthe parameters were set to the default designations (W. R. Pearson etal. (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448). The sequencedatabase searches identified two Incyte Genomics ‘templates’: 252899.6and the potential splice variant 252899.8. It is noted that IncyteGenomics templates are consensus EST sequences that are considered torepresent mRNA transcripts.

Sequence analysis of the BSL2 clone: Incyte Genomics template 252899.8was used to identify Incyte Genomics clone 4616811. Incyte Genomicsclone 4616811 belonged to Incyte Genomics Library ID No. BRAYDIT01,which was originally constructed using poly(A)⁺ RNA from diseasedhypothalamus tissue. Incyte Genomics clone 4616811 was obtained fromIncyte Genomics and used for sequence analysis (ABI cycle sequencer, PEBiosystems) with the primers shown in Table 3. TABLE 3 Clone PrimerSequence SEQ ID NO: 4616811 392.423 GGTGCACAGCTTTGCTGA 34 4616811392.415 GCTGTGCACCAGCTGTTT 35 4616811 392.439 GCTATGAAAGGTCCAGAG 364616811 392.499 GAATCTGGTGGTGTCCAA 37 4616811 392.1716CTCTGTCACCATCACAGG 38 4616811 392.852 CTCTGTCACCATCACACC 39 4616811392.523 GAAATCCCGGATGCTCAC 40 4616811 392.766A ACCACACGTGTTCCAGCA 414616811 392.766B TGCTGGAACACGTGTGGT 42 4616811 392.383GGCCCTCAGCAAAGCTGT 43 4616811 392.1448 AGCTGTAGGTGCCATTCG 44 4616811392.892 AGGGACCTGGACCTCCAC 45 4616811 392.1528 TGGGGGGAATGTCATAGG 464616811 392.1215 AGCAGGCAGGATGACTTA 47 4616811 392.1242AACAGACCACCCACAACC 48 6487516 314.570 GCAAATGGCACCTACAGC 49 6487516314.634 TCTGGGGTGTGATGGTGA 50 6487516 314.450 ATGAAAGGTCCAGAGGGC 516487516 314.584 ACCCATAATTCTTACCCA 52 6487516 314.824 CACAGCTCTGTTTGATCT53 6487516 314.644 CTCCTACCCTCTGGCTGC 54

Notably, the predicted amino acid sequence of Incyte Genomics clone4616811 contained 2 sets of v/c (variable/constant domain) folds,whereas typical B7-related amino acid sequences contain only 1 set ofv/c folds. Seqweb Gap (Genetics Computer Group) analysis indicated thatthe BSL2-4616811 sequence shared less than 50% sequence identity withB7-1, B7-2, BSL1/B7-H1 nucleotide sequences, while the BSL2-4616811amino acid sequence shared less than 35% sequence identity with theB7-1, B7-2, and BSL1/B7-H1 amino acid sequences. A sequence similar toBSL2-4616811 has also been identified as an amyloid precursor proteasein International Patent Application No. WO 00/68266 to G. W. Becker etal.

The nucleotide and predicted amino acid sequences of Incyte Genomicsclone 4616811 (BSL2-4616811) are shown in FIGS. 3A-3B and 3G. Theplasmid carrying DNA encoding BSL2-4616811 (pINCY:BSL2-4616811) wasdeposited with the American Type Culture Collection (ATCC, 10801University Blvd., Manassas, Va. 20110-2209 USA), under ATCC DesignationNo. PTA-1993, on Jun. 6, 2000.

Full-length cloning: To verify the sequence of Incyte Genomics clone4616811, PCR primers were designed to amplify the nucleotide sequencefrom the predicted translation start codon to the predicted translationstop codon of the clone: forward primer BSL2-7 (5′-ATGCTGCGTCGGCG-3′;SEQ ID NO:55); reverse primer BSL2-8 (5′-TCAGGCTATTTCTTGTCCATCATC-3′;SEQ ID NO:56).

A HMVEC library was constructed utilizing the SMART™ RACE cDNAAmplification Kit (CLONTECH) according to manufacturer's instructions,using poly(A)⁺ RNA obtained from human microvascular endothelial cellstreated with TNF-alpha for 1 hr as the RACE reaction template. The PCRmixture included 1 μl PCR-ready HMVEC library, 5 μl PCR buffer(GibcoBRL), 1.5 μl 50 mM MgCl₂, 1 μl 10 mM dNTPs (Boehringer MannheimBiochemicals/Roche Molecular Biochemicals, Indianapolis, Ind.), 25 pMolBSL2-7 primer, 25 pMol BSL2-8 primer, and 1 μl CLONTECH Advantage Enzymemix in a total volume of 50 μl. PCR was performed in a PE BiosystemsThermal Cycler model 9700. The PCR mixture was incubated at 94° C. for 1min, followed by 35 cycles of incubation at 94° C. for 30 sec, 60° C.for 30 sec, and 72° C. for 45 sec, followed by incubation at 72° C. for10 min.

One microliter of the PCR mixture was ligated directly into pCR2.1(Invitrogen) according to the manufacturer's directions. One half of theligation mixture was used for transformation into Max-EfficiencyDH5-alpha E coli cells (GibcoBRL) in accordance with the manufacturer'sdirections. Transformants were plated onto LB agar plates with 100 μg/mlampicillin and 30 μg/ml X-gal and incubated at 37° C. overnight. Whitecolonies were isolated and grown overnight at 37° C. in 5 ml LB brothcontaining 100 μg/ml ampicillin.

Plasmid DNA was isolated from the bacterial culture using Spin Miniprepkit (QIAGEN) according to the manufacturer's directions. DNA wasdigested with EcoRI to release the cloned insert, and the digestionmixture was analyzed by electrophoresis on a 1% agarose gel. Insertfragments larger than 700-bp were sequenced using the vector-specificM13 (5′-GTTTTCCCAGTCACGAC-3′; SEQ ID NO:57) and M13 reverse(5′-CAGGAAACAGCTATGAC-3′; SEQ ID NO:58) sequencing primers (ABI cyclesequencer, PE Applied Biosystems).

Sequence analysis indicated that two splice variants of BSL2 had beencloned: BSL2-L165-21 and BSL2-L165-35b. The BSL2-L165-21 andBSL2-L165-35b splice variants encoded amino acid sequences that eachcontained one v/c fold and were ˜95% identical to one another. SeqwebGap analysis (Genetics Computer Group) also indicated that theBSL2-L165-21 and BSL2-L165-35b nucleotide sequences shared less than 50%sequence identity with B7-1, B7-2, and BSL1/B7-H1 nucleotide sequences,while the BSL2-L165-21 and BSL2-L165-35b amino acid sequences sharedless than 35% sequence identity with the B7-1, B7-2, and BSL-1/B7-H1amino acid sequences.

Sequence analysis further indicated that the amino acid and nucleotidesequences of BSL2-L165-21 shared less than 99% sequence identity withthe PRO352 amino acid and nucleotide sequences, respectively, reportedin International Patent Application No. WO 99/46281 to K. P. Baker etal. The amino acid and nucleotide sequences of BSL2-L165-35b shared lessthan 99.5% sequence identity with the PRO352 amino acid and nucleotidesequences, respectively.

Amino acid sequence alignments using GCG Gap program (GCG, Madison,Wis.) indicated that the longest stretch of identical amino acidresidues shared by BSL2-L165-21 and PRO352 was 88 contiguous amino acidsin length. The longest stretch of identical amino acid residues sharedby BSL2-L165-35b and PRO352 was 168 contiguous amino acids in length.Analysis with ClustalW (J. D. Thompson et al. (1994) Nucleic Acids Res.22:4673-4680 indicated that the longest stretch of identical amino acidsshared by BSL2-4616811 and PRO352 was 206 contiguous amino acids inlength.

Nucleotide sequence alignments indicated that the longest stretch ofidentical bases shared by BSL2-L165-21 and PRO352 was 254 contiguousnucleotides in length. The longest stretch of identical bases shared byBSL2-L165-35b and PRO352 was 305 contiguous nucleotides in length. Thelongest stretch of identical bases shared by BSL2-4616811 and PRO352 was618 contiguous nucleotides in length. Notably, BSL2-L165-35b has alsobeen identified as B7-H3, a co-stimulatory molecule for T-cellactivation (A. I. Chapoval et al. (2001) Nature Immunology 2:269-274).

The nucleotide and predicted amino acid sequences of the BSL2-L165-21splice variant are shown in FIGS. 3C-3D, while the nucleotide andpredicted amino acid sequences of BSL2-L165-35b are shown in FIGS.3E-3F. The plasmid carrying DNA encoding BSL2-L165-21(pCR2.1:BSL2-L165-21) was deposited with the American Type CultureCollection (ATCC, 10801 University Blvd., Manassas, Va. 20110-2209 USA),under ATCC Designation No. PTA-1987, on Jun. 6, 2000. In addition, theplasmid carrying DNA encoding BSL2-L165-35b (pCR2.1:BSL2-L165-35b) wasdeposited with the American Type Culture Collection (ATCC, 10801University Blvd., Manassas, Va. 20110-2209 USA), under ATCC DesignationNo. PTA-1988, on Jun. 6, 2000.

Example 4 Characterization of BSL2

BSL2 expression analysis: BSL2 expression patterns were determined byNorthern blot analysis of various tissue and cell types, using theBSL2-4616811-derived probe that binds to the various forms of BSL2(e.g., BSL2vcvc, BSL2v2c2, and BSL2v1c2). The procedure described forBSL1 was used (see above), and results are shown in FIG. 6B. A 3.6-kbBSL2 mRNA transcript was detected in several cell types. In particular,high levels of BSL2 mRNA were detected in all HMVEC stimulated withTNF-alpha. Moderate levels of BSL2 mRNA were detected in resting THP1cells, and THP1 cells activated with LPS (FIG. 7D). In contrast, lowlevels of BSL2 mRNA were detected in peripheral blood monocytesstimulated with PHA or GM-CSF/IL-4, and BSL2 mRNA was not detected inresting or stimulated peripheral blood T-cells, or in resting RAJIcells, resting RAMOS cells, or serum starved H292 cells (FIG. 7D).

PCR assay to determine relative abundance of BSL2-4616811 (BSL2vcvc) andBSL2-L165-35b (BSL2v1c2): To determine whether BSL2-4616811 (BSL2vcvc)or BSL2-L165-35b (BSL2v1c2) was predominant species of BSL2, and whetherpredominance corresponded with cell type and/or stimulus type, thefollowing experimental approach was used. Analysis of the genomicsequence of BSL2 indicated that the sequence includes several exonsseparated by introns. It was presumed that a primary transcript wasproduced from this sequence, and the primary transcript was spliced toyield BSL2-4616811 mature RNA. Analysis of BSL2-4616811 sequence showedthat it coded for the following: a 5′ UTR, an initiating ATG, a signalpeptide sequence, a variable Ig fold (v1), an Ig constant fold (c1), anIg V fold (v2), an Ig C fold (c2), a short hinge a putativetransmembrane domain, a short cytoplasmic tail, a stop codon, and a 3′UTR.

The BSL2-4616811 coding sequence appeared unique in the human genome, asthe v1 and c1 (v1c1) sequence was about 95% identical to the v2 and c2(v2c2) sequence at the amino acid level. Importantly, all of thestructurally important residues were conserved in the v1c1 and v2c2amino acid sequences, and most of the changes from v1c1 to v2c2 wereconservative changes. A schematic of the BSL2-4616811 (BSL2vcvc),BSL2-L165-21 (BSL2v2c2), and BSL2-L165-35b (BSL2v1c2) v/c domains isshow in FIG. 3H.

Despite the 96% identity between v1c1 and v2c2 at the nucleotide level,sequence comparison between the two regions using GCG Gap revealed oneshort region of relatively low homology. A forward primer designatedBSL2-9 was designed to take advantage of this short region of lowhomology. BSL2-9 was designed to bind specifically to v1 (FIG. 8Edemonstrates the specificity of the BSL2-9 primer). A reverse primer,BSL2-11 was designed to hybridize to the hinge sequence.

Notably, the BSL2-4616811 transcript contained both the BSL2-9v1-binding site in v1 and the homologous site in v2. The BSL2-L165-35btranscript contained only the BSL2-9 v1 binding site. The BSL2-L165-21transcript contained only the v2 site. Accordingly, PCR performed withprimers BSL2-9 and BSL2-11 was expected to produce: 1) a PCR product ofapproximately 1150-bp, representing BSL2-4616811; or 2) a PCR product ofabout 550-bp, representing BSL2-L165-35b.

PCR was initially conducted with BSL2-4616811 plasmid to confirm thespecificity of the BSL2-9 primer. PCR was performed using 2 μl of 10ng/μl BSL2-4616811 plasmid DNA, 5 μl PCR buffer (GibcoBRL), 1.5 μl of 25mM MgCl₂ (GibcoBRL), 1 μl of 10 mM dNTPs (GibcoBRL), 2.5 μl of 10pMol/μl BSL2-9 primer (5′ TGGTGCACAGCTTTGCT 3′; SEQ ID NO:59), 2.5 μl of10 pMol/μl BSL2-11 primer (5′ TCTGGGGGGAATGTCAT 3′; SEQ ID NO:60), 0.5μl of 5 U/μl GibcoBRL platinum Taq DNA polymerase (Cat. # 10966-018),and 35 μl dH₂O. PCR was performed on a PE Biosystems 9700 using thefollowing cycling conditions: 94° C. for 30 sec; followed by 35 cyclesof 94° C. for 30 sec, 61° C. for 30 sec, 72° C. for 60 sec; followed by72° C. for 10 min. Following this, 40 μl of the PCR reaction was run ona 1.2% agarose gel next to Lambda BstEII DNA ladder (New England BiolabsBeverly, Mass.; Cat. # 301-4S). The 1150-bp band was predominant,indicating that the BSL2-9 PCR primer was specific for the BSL2-4616811sequence (FIG. 8E).

RAJI, RAMOS, PM-LCL, PL-LCL, and CE-LCL B-like cell lines; HL-60 andThp1 monocytic like cell lines; and CEM and Hut78 T-like cell lines weregrown in RPMI 1640 with 10% FBS and 1% GibcoBRL penicillin/streptomycinto a concentration of 5×10⁵ cells/ml. The cultures were split andone-half of the B-like and T-like cell cultures were stimulated with 30ng/ml PMA and 1 μM ionomycin for 24 hr. Monocytic cell lines werestimulated with 1 μg/ml LPS for 24 hr. Early passage HUVEC were grown to90% confluence, and one-half of the culture was stimulated with 10 ng/mlTNFα, and harvested at 6 or 24 hr. Unstimulated HUVEC were harvested at24 hr.

Peripheral blood T-cells from two donors were purified as described,above. Cells were added (1×10⁶ cells/ml) to RPMI 1640 with 10% humanserum and 1% GibcoBRL penicillin/streptomycin. Cells were then incubatedat 37° C. in 5% CO₂ for 72 hr. T75 flasks were coated with 1 μg/ml CD3MAb G19.4 (described herein) in PBS for 4 hr at 37° C. The flask waswashed twice with PBS, and cells were added (1×10⁵ cells/ml) in RPMI1640 with 10% human serum and 1% GibcoBRL penicillin/streptomycin. CD28MAb 9.3 (described herein) was added to a final concentration of 1μg/ml. Cells were grown at 37° C. in 5% CO₂ for 72 hr. Cells were added(1×10⁶ cells/ml) to RPMI 1640 with 10% human serum and 1% GibcoBRLpenicillin/streptomycin. PMA was added to 30 ng/ml and ionomycin wasadded to 1 μM and incubated at 37° C. in 5% CO₂ for 48 hr. Proliferationof stimulated T-cells was confirmed visually. All cell types werepelleted and frozen on dry ice and stored at −70° C. until use.

Total RNA was prepared using the Invitrogen (Carlsbad, Calif.) SNAPtotal RNA isolation kit (Cat. # K1950-01) according to themanufacturer's instructions. First strand cDNA was produced using theGibcoBRL Superscript First-Strand Synthesis System for RT-PCR (Cat. #11904-018) according to the manufacturer's instructions for oligo dTpriming. PCR was performed using 2 μl first strand cDNA, 5 μl PCR buffer(GibcoBRL), 1.5 μl of 25 mM MgCl₂ (GibcoBRL), 1 μl of 10 mM dNTPs(GibcoBRL), 2.5 μl of 10 pMol/μl BSL2-9 primer (5′ TGGTGCACAGCTTTGCT 3′;SEQ ID NO:61), 2.5 μl of 10 pMol/μl BSL2-11 primer (5′ TCTGGGGGGAATGTCAT3′; SEQ ID NO:62), 0.5 μl of 5 U/μl GibcoBRL platinum Taq DNA polymerase(Cat. # 10966-018), and 35 μl dH₂O. PCR was performed on a PE Biosystems9700 using the following cycling conditions: 94° C. for 30 sec; followedby 35 cycles of 94° C. for 30 sec, 61° C. for 30 sec, 72° C. for 60 sec;followed by 72° C. for 10 min. Following this, 40 μl of the PCR reactionmixture was run on a 1.2% agarose gel.

PCR analysis indicated that certain cell types contained predominantlythe BSL2-4616811 (BSL2vcvc) transcript, with or without stimulation.Unstimulated and stimulated PL-LCL cells showed higher levels of theBSL2-4616811 transcript than the than the BSL2-L165-35b transcript (FIG.8A). Both unstimulated and stimulated HUVEC cells showed higher levelsof the BSL2-4616811 transcript than the BSL2-L165-35b transcript (FIG.8B).

In contrast, other cell types contained predominantly the BSL2-L165-35b(BSL2v1c2) transcript, with or without stimulation. Stimulated RAJIcells, and unstimulated and stimulated RAMOS cells showed higher levelsof the BSL2-L165-35b transcript than the BSL2-4616811 transcript (FIG.8A). Unstimulated and stimulated HL60 cells showed higher levels of theBSL2-L165-35b transcript than the BSL2-4616811 transcript (FIG. 8B).

In addition, certain cell types showed an increase in BSL2-4616811(BSL2vcvc) transcript levels upon activation. Unstimulated PM-LCL cellsshowed higher levels of the BSL2-4616811 transcript, which increasedrelative to the BSL2-L165-35b transcript upon stimulation (FIG. 8A).Similarly, unstimulated CE-LCL cells showed higher levels of theBSL2-4616811 transcript, which increased relative to the BSL2-L165-35btranscript upon stimulation (FIG. 8B). Unstimulated Thp1 cells showedequivalent levels of the BSL2-4616811 and the BSL2-L165-35b transcript,however, levels of the BSL2-4616811 transcript increased uponstimulation (FIG. 8B). Unstimulated peripheral blood T-cells from donor079 showed predominantly BSL-L165-35b but shifted to predominantlyBSL2-4616811 upon stimulation. Peripheral blood T-cells from donor 124showed a less dramatic shift from BSL2-L165-35b to BSL2-4616811 uponstimulation (FIG. 8C). Unstimulated CEM cells showed higher levels ofthe BSL2-L165-35b transcript, but levels of the BSL2-4616811 transcriptincreased upon activation (FIG. 8D).

Other cell types showed an increase in BSL2-L165-35b (BSL2v1c2) levelsupon activation. Unstimulated HUT78 cells showed higher levels of theBSL2-4616811 transcript, but levels of the BSL2-L165-35b transcriptincreased upon activation (FIG. 8D). These results, coupled with theconservation of the amino acid sequences in all four Ig folds, theconservation of structurally important amino acid residues in all fourIg folds, and the conservative nature of the amino acid differencesbetween v1c1 and v2c2, support a function for BSL2-4616811 (BSL2vcvc)which is distinct from BSL2-L165-35b (BSL2v1c2) and BSL2-L165-21(BSL2v2c2).

To rule out the presence of PCR artifacts, the PCR product from HUVECactivated with TNFα for 24 hr was cloned using the Invitrogen OriginalTA Cloning Kit (Cat. # K2000-01) according to the manufacturer'sdirections. The construct was used for transformation into bacterialcells, and 25 white colonies were isolated and grown in LB with 100μg/ml ampicillin. DNA preps were made and sequencing was performed. Ofthe 25 clones, 10 clones did not contain insert, 3 clones did notproduce readable sequence, 3 clones contained BSL2-related sequenceswith extensive deletions, and the remaining 9 clones produced sequenceconsistent with BSL2-4616811.

It was noted that no PCR product corresponding to BSL2-4616811 orBSL2-L165-35b was observed in unstimulated RAJI cells. To confirm thisresult, PCR was repeated for unstimulated and unstimulated RAJI cells aspreviously described. Again, no PCR product was detected for theunstimulated RAJI cells. Following this, PCR was performed using G3PDHprimers with template isolated from unstimulated and stimulated RAJIcells. Cycling conditions were identical to those described above,except the annealing temperature was 55° C., and the extension time was45 sec. Relatively equal amounts of G3PDH product was detected for bothunstimulated and stimulated RAJI cells. This provided further supportfor the observation that unstimulated RAJI cells do not containdetectable BSL2 transcript.

BSL2-L165-35b-Ig (BSL2v1c2-Ig) fusion construct: BSL2-L165-35b-Ig(BSL2v1c2-Ig) was constructed as follows. The extracellular domain ofBSL2-L165-35b was amplified by PCR from an L165-35b DNA preparation. ThePCR reaction included 1 μl of a 1/1000 dilution of the L165-35btemplate, PCR buffer, dNTP, and MgCl₂ from Gibco (described herein), 2.5μl of 10 μM primer BSL2-L165-21-Ig-1, 2.5 μl of 10 μM primerBSL2-L165-21-Ig-2 (both primers were described for the BSL2-4616811-Igconstruction, below), and 1 μl CLONTECH AdvanTaq Plus (Palo Alto,Calif.; Cat. # 8431-1) in a total volume of 50 μl. PCR conditionsincluded incubation at 94° C. for 1 min; followed by 20 cycles ofincubation at 94° C. for 30 sec, 59° C. for 30 sec, 72° C. for 45 sec;followed by incubation at 72° C. for 10 min. PCR was performed on a PEApplied Biosystems (Perkin-Elmer, Foster City, Calif.) GeneAmp PCRSystem 9700 thermal cycler.

Following PCR, 10 μl of the reaction mixture was run on a 1.2%agarose/0.5×TBE gel to check for product. The remainder of the PCRreaction mixture was digested at 37° C. with 20 U KpnI (Roche Molecular,Indianapolis Ind.; Cat. # 899 186) and 20 U EcoRI (Roche Molecular, Cat.# 703 737) in a total volume of 80 μl for 16 hr. The digestion mixturewas run on a 1.2% agarose/0.5% TBE gel. A predominant band of about700-bp was purified using the QIAGEN Qiaquick Gel Extraction Kit(Valencia Calif.; Cat. # 28704). Following this, 5 μl of 50 μl recoveredproduct was run on a 1.2% agarose/0.5% TBE gel and the DNA concentrationwas estimated as 10 ng/μl. Then, 15 ng recovered product was ligatedinto 30 ng of L200-1 (vector described herein) digested with KpnI plusEcoRI using 5 U Gibco T4 DNA ligase (Cat. # 15224-041) in a total volumeof 10 μl (ligation number L315). The reaction was incubated at 14° C.for 3 hr.

One-half of the reaction mixture was used for transformation into GibcoMaxEfficiency DH5α competent E coli (Cat. # 18256-012). Transformantswere plated on LB plates plus 100 μg/ml ampicillin and incubated at 37°C. for 18 hr. Bacterial colonies were grown in 5 ml LB plus 100 μl/mlampicillin for 16 hr. DNA preps were made using QIAGEN Qiaprep SpinMiniprep Kit (Cat. # 27106). Isolates L315-1 and L315-3 were determinedby sequence analysis to be correct. The BSL2-L165-35b-Ig fusion wasproduced by transient transfection of COS as described forBSL2-4616811-Ig, below. BSL2-L165-35b-Ig fusion protein was purifiedusing methods described herein. The concentration of theBSL2-L165-35b-Ig fusion protein was estimated by absorbance at 280 nmassuming an extinction coefficient of 1.4. The nucleotide and predictedamino acid sequence of BSL2-L165-35b-Ig (BLS2v1c2-Ig) is shown in FIGS.4C-4D.

BSL2-L165-21-Ig (BSL2v2c2-Ig) fusion construct: PCR amplification of theBLS2-L165-21 cDNA template was performed using BSL2-L165-21-Ig-1 andBSL2-L165-21-Ig-2 primers (both primers described for theBSL2-4616811-Ig construction, below). The PCR reaction mixture included42 μl GibcoBRL PCR Super Mix; 2 μl template (approximately 300 ng/μl;diluted 1:100); 2.5 μl of 1 pM/μl BSL2 L165-21 Ig-1 primer; 2.5 μl of 1pM/μl BSL2 L165-21 Ig-2 primer; and 1 μl CLONTECH Advantage 2 DNAPolymerase mix. The PCR reaction was carried out as follows. Thereaction mixture was incubated at 95° C. for 5 min, followed by 35cycles of incubation at 95° C. for 45 sec, 58.5° C. for 45 sec, and 72°C. for 90 sec, followed by incubation at 72° C. for 5 min. The reactionmixture was then held at 4° C.

Following this, the PCR product was cloned into a TA vector (Invitrogen;Cat. # K2000-01 and K2000-40) using the protocol provided by themanufacturer. The ligation reaction included 5 μl H₂O; 1 μl 10× buffer(Roche, Mannheim, Germany); 2 μl vector pCR2.1; 1 μl PCR product(unpurified); and 1 μl T4 Ligase (Roche). One-half of the ligationmixture was used for transformation into max efficiency DH5α competentcells (Invitrogen) and plated as described, above. White colonies wereisolated and grown in culture, and minipreps were performed (QIAGEN).The miniprep DNA and vector L200-1 (described herein) were digested withKpnI and EcoRI in SuRE/Cut Buffer A (Boehringer Mannheim, Mannheim,Germany). The digestion mixture included 14 μl miniprep DNA or L200-1vector; 2 μl KpnI; 2 μl EcoRI; 2 μl 10× Buffer A; and 10 μl dH₂O. Thedigestion mixture was electrophoresed on a 1% SeaPlaque Low Melt agarosegel (FMC, Rockland, Me.). Bands of approximately 750-bp from theminiprep DNA and approximately 6200-bp from the L200-1 vector wereexcised and melted at 65° C.

Next, ligation reactions were carried out in 10 μl total volume. Theligation reactions included reagents listed in the table below. 5:1 2:1Insert:Vector Insert:Vector Control Insert BSL2-L165-21 5 μl 2 μl 0 μlVector L200-1 1 μl 1 μl 1 μl T4 Ligase (Roche) 1 μl 1 μl 1 μl 10 ×Ligation Buffer (Roche) 1 μl 1 μl 1 μl dH₂O 2 μl 5 μl 7 μl

Ligation reactions were incubated overnight at 14° C. After this, theligation mixture was used for transformation into DH5α Max EfficiencyCells (Invitrogen). For transformations, 100 μl of cells were aliquottedinto pre-chilled 14 ml snap cap tubes (Falcon, Becton Dickinson,Franklin Lakes, N.J.). Next, 2 μl of DNA was added to the cells, andcells were incubated on ice for 25-30 min. Cells were heat-shocked at42° C. for 45 sec, and then placed back on ice for at least 2 min.Following this, 900 μl of SOC growth medium (GibcoBRL) was added to eachtube. The tubes were incubated for 1 hr at 37° C. After this, the cellswere plated onto LB plates supplemented with 100 μg/ml ampicillin.Plates were then incubated at 37° C. overnight.

Individual colonies were isolated and grown in 3 ml of LB growth mediumsupplemented with 100 μg/ml ampicillin. An initial miniprep wasperformed using a commercially available kit (QIAGEN, Valencia, Calif.;Cat. # 27106) to confirm insert orientation and sequence quality. Allminiprep samples tested were shown to contain the correct insertorientation. After testing for sequence quality, a Plasmid Giga Prep kit(QIAGEN; Cat. # 12191) was used for large-scale production of DNA. Forthe Giga Prep, 2.5 L of culture was divided into three flasks. Theseflasks were incubated approximately 15 hr, but not longer 18 hr.Following this, a Giga Prep was performed according to themanufacturer's directions. The nucleotide and predicted amino acidsequence of BSL2-L165-21-Ig (BLS2v2c2-Ig) is shown in FIGS. 4E-4F.

BSL2-4616811-Ig (BSL2vcvc-Ig) fusion construct: To construct theBSL2-4616811-Ig (BSL2vcvc-Ig) plasmid, the BSL2-4616811 extracellulardomain was PCR amplified from first strand cDNA (GibcoBRL Cat. #11904-018). cDNA was prepared from RNA purified from THP1 cellsstimulated with 100 ng/ml LPS for 2 hr. RNA was purified usingInvitrogen FastTrack 2.0 (Cat. # K1593-02). The PCR reaction included 1μl cDNA; 5 μl GibcoBRL 10×PCR buffer; 1.5 μl of 25 mM MgCl₂; 1 μl of 10mM dNTPs (Boehringer-Mannheim); 2.5 μl BSL2-L165-21-Ig-1 primer (10pM/μl); 2.5 μl BSL2-L165-21-Ig-2 primer (10 pM/μl); 1 μl CLONTECHAdvantage polymerase (Cat. # 8417-1); and 35.5 μl milliQ H₂O. Theprimers contained the following sequences: BSL2-L165-21-Ig-1: 5′ gg ggtacc ATG CTG CGT CGG CG 3′ (SEQ ID NO:63); and BSL2-L165-21-Ig-2: 5′ cggaa ttc TGG GGG GAA TGT CAT AG 3′ (SEQ ID NO:64). PCR samples wereincubated at 94° C. for 1 min; followed by 35 cycles of incubation at94° C. for 30 sec, 59° C. for 30 sec, and 72° C. for 45 sec; followed byincubation at 72° C. for 10 min.

Following this, 30 μl of the PCR reaction was run on a 1.2%agarose/0.5×TBE gel. A band of approximately 1100-bp was excised andpurified using QIAGEN Gel Extraction Kit (Cat. # 28704). One microliterof the purified PCR product (L254) was ligated into pCR2.1 using the TAcloning kit (Invitrogen; Cat. # K2050-01). Five microliters of theligation mixture was used for transformation into MAX EfficiencyDH5-alpha competent bacteria (GibcoBRL; Cat. # 18258-012), andtransformants were plated onto LB plates containing 100 μg/ml ampicillinand 800 μg X-Gal. White colonies were inoculated into 5 ml LB brothcontaining 100 μg/ml ampicillin, and grown at 37° C. for 18 hr. PlasmidDNA was purified with QIAGEN spin miniprep kit (Cat. # 27106). PlasmidDNA was digested with KpnI and EcoRI. Plasmids containing inserts ofabout 1300-bp were sequenced using Applied Biosystems automated DNAsequencers (ABI 3700 capillary array sequencers). L254-7 was determinedto contain wild-type BSL2-4616811 sequence.

The Fc portion of human IgG1 was PCR amplified using 0.001 μl BSL1-Ig,described herein; 5 μl GibcoBRL PCR buffer; 1.5 μl of 25 mM MgCl₂; 1 μlof 10 mM dNTPs (Boehringer-Mannheim); 2.5 μl Ig-1 primer (10 pM/μl) 2.5μl BSL1Ig-2 primer (10 pM/μl); 1 μl CLONTECH Advantage polymerase; and36 μl dH₂O. The primers contained the following sequences: IgG-1: 5′ggaa ttc GAG CCC AAA TCT TGT GAC AA 3′ (SEQ ID NO:65); and BSL1Ig-2 gc gctct aga TCA TTT ACC CGG AGA CAG G (SEQ ID NO:66). PCR samples wereincubated at 94° C. for 1 min; followed by 25 cycles of incubation at94° C. for 30 sec, 59° C. for 30 sec, and 72° C. for 30 sec; followed byincubation at 72° C. for 10 min.

Following this, 30 μl of the PCR reaction was run on a 1.2%agarose/0.5×TBE gel. A band of about 700-bp was excised and purifiedusing QIAGEN Qiaquick gel extraction kit. Two microliters of thepurified fragment (L174) was ligated into pCR2.1 using the TA cloningkit (Invitrogen). Five microliters of the ligation mixture was used fortransformation into GibcoBRL MAX Efficiency DH5-alpha competentbacteria, and transformants were plated onto LB plates containing 100μg/ml ampicillin and 800 μg X-gal. Plates were incubated for 18 hr at37° C. White colonies were inoculated into 5 ml LB broth containing 100μg/ml ampicillin and grown at 37° C. for 18 hr. Plasmid DNA was preparedusing QIAGEN Qiaprep spin miniprep kit. Plasmid DNA was digested withEcoRI and run on an agarose gel. Plasmids containing inserts of about900-bp were sequenced. L174-3 was determined to contain wild-type humanIgG1 Fc sequence.

L174-3 was digested with EcoRI/XbaI and separated on a 1.2%agarose/0.5×TBE gel. A band of about 750-bp was excised and purifiedusing QIAGEN gel extraction kit. Ten microliters of the purifiedfragment was run on an agarose gel next to a standard to obtain anestimate of the concentration. Approximately 20 ng of the EcoRI/XbaIfragment was ligated (Ligation 200) into 40 ng of EcoRI/XbaI digestedpcDNA3.1+vector (Invitrogen) using GibcoBRL high concentration T4 DNAligase (5 U/μl) diluted in GibcoBRL T4 DNA ligase buffer. Fivemicroliters of the ligation mixture was used for transformation into MAXEfficiency DH5-alpha competent bacteria (GibcoBRL), and transformantswere plated onto LB plates containing 100 μg/ml ampicillin. Plates wereincubated at 37° C. for 18 hr. Colonies were inoculated into LB brothcontaining 100 μg/ml ampicillin. Plasmid DNA was purified using QIAGENspin miniprep kit and sequenced. The L200-1 sequence was determined tobe identical to the L174-3 sequence.

The L254-7 BSL2-4146811 construct was digested with KpnI/EcoRI. A bandof about 1300-bp was excised from a 1.2% agarose/0.5×TBE gel and ligatedinto the L200-1 Fc construct digested with KpnI/EcoRI. Five microlitersof the ligation reaction was used for transformation into MAX EfficiencyDH5-alpha cells and plated onto LB plates containing 100 μg/mlampicillin. Colonies were grown, and plasmid DNA was purified as above.Plasmid DNA was digested with KpnI/XbaI and separated on an agarose gelas above. Plasmids containing a band of about 2-kb were sequenced asabove. BSL2-4616811-Ig was determined to contain the wild-typeBSL2-4616811 and wild-type human IgG1 sequences. The nucleotide andpredicted amino acid sequence of BSL2-4616811-Ig (BSL2vcvc-Ig) is shownin FIGS. 4A-4B. Plasmids comprising BSL2vcvc-Ig, BSL2v1c2-Ig, orBSL2v2c2-Ig sequences were deposited as a mixture with the American TypeCulture Collection (ATCC, 10801 University Blvd., Manassas, Va.20110-2209 USA), under ATCC Designation No. ______, on Feb. 8, 2002.

BSL2-4616811-Ig (BSL2vcvc-Ig) and BSL2-L165-35b (BSL2v1c2) constructionfor expression in CHO cells: The extracellular domain of human BSL2containing either a vc (BSL2v1c2) or vcvc (BSL2vcvc) region was PCRamplified from cDNA and cloned into mammalian expression vector pD16(described in U.S. Pat. No. 6,051,228) using established methods(described by E. Kondri et al. (1997) BioTechniques 23(5): 830-833). Theprimers included a vcvc forward primer (5′ ACT ATA GGG AGA CCC AAG CTTGGT ACC GGA TCC ATG CTG CGT CGG CGG GGC AGC CCT GGC 3′; SEQ ID NO:136);vcvc and v1c2 reverse primer (5′ GTC ACA AGA TTT GGG CTC CGG ATC CTC TGGGGG GAA TGT CAT AGG CTG CCC 3′; SEQ ID NO:137), and v1c2 forward primer(5′ CAA GCT TGG TAC CGG ATC CAT GGA AGC CCC AGC TCA GCT TCT CTT CCT CCTGCT ACT CTG GCT CCC AGA TAC CAC CGG AAC AGG AGC CCT GGA GGT CCA G 3′;SEQ ID NO:138). The CHO vcvc construct incorporated the native signalpeptide sequence and the CHO v1c2 construct incorporated the cd40 signalpeptide sequence to direct the secretion of protein from mammaliancells. In addition, a stop codon was added to the end of the Ig sequenceusing a PCR primer.

The vector backbone was derived from the Invitrogen plasmid pcDNA3 andcontained the following modifications. The neomycin resistance gene frompcDNA3 was replaced with the dihydrofolate reductase (DHFR) undercontrol of the SV40 promoter missing the enhancer (also referred to as“weakened DHFR”). The SV40 promoter contained the SV40 origin ofreplication, so the vector could be used for transient expression ofprotein. The CMV promoter was used to express the fusion of interest,and the polyadenylation signal was obtained from the bovine growthhormone gene. The expression cassette for the fusion of interest wasflanked by transcription termination sequences (i.e. 5′ to the promoterand 3′ to the poly(A)⁺ site). The ampicillin resistance gene and ColE1origin was included to allow plasmid propagation in E. coli. All DNAfragments for cloning purposes were prepared by using standard molecularcloning methodologies (J. Sambrook (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratories Press, Cold SpringHarbor, N.Y.). The coding region was confirmed by DNA sequence analysis.

BSL2-Ig (BSL2vcvc-Ig or BSL2v1c2-Ig)-producing cell lines were generatedby transfecting CHO DG44 cells (G. Urlaub et al. (1986) Somat. Cell.Mol. Genet. 12(6):555-66) with an expression vector (pD16.hBSL2.Ig;either BSL2vcvc-Ig or BSL2v1c2-Ig) using a modified method of the highcopy electroporation (J. Barsoum (1990) DNA and Cell Biol. 9:293-300;U.S. Pat. No. 4,956,288). For electroporation, 200 μg of vector(pD16.hBSL2.Ig; either BSL2vcvc-Ig or BSL2v1c2-Ig) and sheared herringsperm DNA (as carrier) were co-precipitated with ethanol and resuspendedin sterile PF CHO protein-free medium (JRH Biosciences, Lenexa, Kans.)CHO DG44 cells were obtained in exponential growth phase, and PF CHO wasused as the electroporation medium. Cells were electroporated at 300volts, 960° F. in a Bio-Rad gene pulser. Serum was omitted at all timesduring the process of cell line generation.

Following electroporation, cells were allowed to recover one day innon-selective media (PF CHO with 10 μg/ml recombinant insulin, 4 mML-glutamine, 4 μg/ml hypoxantine, and 0.72 μg/ml thymidine). Cells werethen seeded in 96-well plates at 250 or 1000 cells/well in selectivemedia. Selective media was PF CHO with 10 μg/ml recombulin (recombinantinsulin; GibcoBRL, Cat. # 28150-019), 4 mM L-glutamine, and 50 or 100 nMMTX (methotrexate). The plates were screened two weekspost-electroporation by ELISA for the selection of masterwells producingthe highest level of BSL2-Ig (BSL2vcvc-Ig or BSL2v1c2-Ig) fusionprotein. As used herein, a “masterwell” is a well containing transfectedcells that have not been clonally isolated.

The cell lines (10 masterwells) expressing the highest levels of BSL2-Ig(BSL2vcvc-Ig or BSL2v1c2-Ig) were selected for further amplification.The cell lines were passed once per week through increasingconcentrations of MTX (50 nM->100 nM->250 nM->500 nm). T-flasks wereseeded at 2-5×10⁴ cells/ml, depending on the cells' tolerance to theprior MTX level. Amplification was assessed by comparing titers ofamplified and non-amplified masterwells in 7-day expression assaysperformed in 12-well tissue culture plates. Protein expression levelswere assayed by enzyme linked immunosorbant assay (ELISA) and confirmedby SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis).

Fusion protein (BSL2vcvc-Ig or BSL2v1c2-Ig) was isolated by affinitypurification using a protein A column. The protein was eluted from thecolumn by five volumes of 0.1 M citrate pH 3, into one volume Tris pH 3.The eluted protein solution was dialyzed against PBS. To freezetransfected CHO cells, the culture was frozen while in exponentialgrowth phase, as established by cell counts. The cultures showed highviability levels (>9% by trypan blue exclusion).

BSL2 monoclonal antibodies: The BSL2-4616811-Ig (BSL2vcvc-Ig) fusionprotein was purified by affinity purification following expression inCOS cells as described for BSL3-Ig, below. It is noted that theBSL2-4616811-Ig fusion protein used in the experiments that follow wasisolated from COS cells, unless indicated otherwise. The purifiedBSL2-4616811-Ig fusion protein was used to immunize mice using theprotocol described for BSL3-Ig, except that a fourth boost was used.Following this, hybridoma cell lines were constructed and anti-BSL2 MAbswere isolated as described for BSL3, below. Hybridoma cells producinganti-BSL2-1F7G2 MAb, anti-BLS2-3E6D3 MAb, anti-BSL2-4C2C6 MAb, oranti-BLS2-5D7E2 MAb were deposited with the American Type CultureCollection (ATCC, 10801 University Blvd., Manassas, Va. 20110-2209 USA),under ATCC Designation No. ______, ______, ______, and ______,respectively, on Feb. 8, 2002.

Example 5 Identification of BSL3

Database searches: BSL3 was identified by BLAST and FASTA analysis ofthe Incyte Genomics sequence databases utilizing the B7-1 or B7-2proteins as query sequences, and the parameters described for the BSL2searches. The sequence database searches identified Incyte Genomics‘gene’ 117327. It is noted that an Incyte Genomics gene is an ESTsequence that is grouped with similar sequences, and considered torepresent the product of a single genomic locus. The Incyte Genomicsgene 17327 has since been renamed as Incyte Genomics gene 899898. In asecondary screen, the BLAST and FASTA programs were used to search theIncyte Genomics sequence databases for sequences related to the mouseAF142780 gene (potential orthologue of the 117327 gene), using thepreviously described search parameters. These searches identified IncyteGenomics gene 143522.

The 143522 and 117327 genes were then used to identify Incyte Genomicsclones 3844031 and 3207811, respectively. The 3844031 clone belongs toIncyte Genomics Library ID No. DENDTNT01, which was originallyconstructed utilizing poly(A)⁺ RNA isolated from untreated dendriticcells from peripheral blood. The 3207811 clone belongs to IncyteGenomics Library ID No. PENCNOT03, which was originally constructedutilizing poly(A)⁺ RNA isolated from corpus cavernosum tissue. The3844031 and 3207811 clones were obtained from Incyte Genomics, andsequenced (ABI cycle sequencer, PE Biosystems) using the primers shownin Table 4. TABLE 4 Clone Primer Sequence SEQ ID NO: 3844031 316.491GAAGGCCTCTACCAGGTC 67 3844031 316.512 CTTTAGGCGCAGAACACT 68 3844031316.203 AAGGGTCAGCTAATGCTC 69 3844031 316.379 TCAGTTTGCACATCTGTA 703844031 316.1538 TATGCTATCAAGATTCCA 71 3844031 316.1839GTAAAGTGCAGTAGTGCT 72 3844031 316.1601 TATGAGCTCACAGACAGG 73 3207811315.560 AGGTTCAGATAGCACTGT 74 3207811 315.468 ACTTATCTGAAATTGCTG 753207811 315.493 TTGATATGCTCATACGTT 76 3207811 315.1011GAATTCTGTGGGTCCAGG 77 3207811 315.601 CATGTTTAATGGTGGTTT 78 3207811315.498 AAAGCTGTATTCTTCAAA 79

Full-length cloning of BSL3: To clone the 5′ end of BSL3, the SMART™RACE cDNA Amplification kit (CLONTECH) was used according to themanufacturer's directions. The 5′ RACE library was constructed using 1.0μg of poly(A)⁺ RNA template obtained from human microvascularendothelial cell treated with TNF-alpha for 1 hr. The 5′ RACE reactionmixture contained 2 μl RACE-ready cDNA, 1×PCR buffer (GibcoBRL), 200 μMdNTP (Boehringer Mannheim), 1.5 mM MgCl₂, 1 μl CLONTECH Advantage enzymemix, 1× CLONTECH SMART primer, and 25 pMol BSL3 3′ specific primer(BSL3-2 5′-GAACACTGGTGACCTGGTAGAG-3′; SEQ ID NO:80) in a total volume of50 μl. The 5′ RACE reaction was performed in a GeneAmp PCR System 9700machine (PE Applied Biosystems) using an initial denaturation step ofincubation at 94° C. for 1 min; followed by 35 cycles of incubation at94° C. for 30 sec, 62° C. for 30 sec, 72° C. for 30 sec; followed byincubation at 72° C. for 10 min.

The PCR products were analyzed by gel electrophoresis using a 1.2%agarose gel (GibcoBRL) with 0.5×TBE and 10 μg/ml ethidium bromide(Bio-Rad). An ˜875-bp fragment was excised from the gel and purifiedusing the QIAGEN Qiaquick Gel Extraction kit according to themanufacturers directions. One microliter of the purified fragment wasmixed with 2 μl pCR2.1 pTADV cloning vector (CLONTECH), 2 μl ligationcocktail (GibcoBRL), and 4 U T4 DNA ligase (GibcoBRL) in a total volumeof 10 μl, and the ligation mixture was incubated at 14° C. for 4 hr.Five microliters of the ligation mixture was used for transformationinto Max-Efficiency DH5-alpha E. coli cells (GibcoBRL) according to themanufacturers directions, and transformants were plated onto LB platescontaining 100 μg/ml ampicillin and 30 μg/ml X-gal. White colonies werepicked and grown in 5 ml of LB broth containing 100 μg/ml ampicillin.DNA was purified from the bacteria using the Qiaprep Spin Miniprep Kit(QIAGEN). Plasmid DNA was digested with EcoRI and analyzed by agarosegel electrophoresis. Plasmid isolates containing an EcoRI fragment ofapproximately 900-bp were retained for sequencing (ABI cycle sequencer,PE Biosystems). The sequence was analyzed by Seqweb Gap (GeneticsComputer Group).

The 5′ RACE library was then used as a template for PCR amplification.The PCR mixture contained 1 μg 5′ RACE library template, 1×PCR buffer(GibcoBRL), 200 μM dNTP (Boehringer Mannheim), 1.5 mM MgCl₂, 1 μlCLONTECH Advantage enzyme mix, 25 pMol forward primer (BSL3-3:5′-CCGGGGTACCATGATCTTCCTCCTGCTAATGTTG-3′; SEQ ID NO:81), and 25 pMolreverse primer (BSL3-4: 5′-GCGCTCTAGATCAGATAGCACTGTTCACTTCCC-3′; SEQ IDNO:82) in a total volume of 50 μl. PCR was performed in a GeneAmp PCRSystem 9700 (PE Applied Biosystems) machine using an initialdenaturation step of incubation at 94° C. for 1 min; followed by 30cycles of incubation at 94° C. for 30 sec, 60° C. for 30 sec, 72° C. for30 sec; followed by incubation at 72° C. for 10 min.

An ˜800-bp BSL3 PCR product was obtained. To clone the BSL3 fragment, 1μl of the PCR product was mixed with 2 μl pCR2.1 cloning vector(Invitrogen), 2 μl ligation buffer (GibcoBRL), 4 U T4 DNA ligase(GibcoBRL), and 4 μl H₂O. The ligation mixture was incubated at 14° C.for 4 hr, and 5 μl microliters of the mixture was used to transfectMax-Efficiency DH5-alpha E. coli cells (GibcoBRL). Transformants wereplated onto LB agar plates containing 100 μg/ml ampicillin and 30 μg/mlX-gal. Eighteen white colonies were isolated and grown in 5 ml of LBbroth containing 100 μg/ml of ampicillin. DNA was purified from thebacterial culture using the Qiaprep Spin Miniprep Kit (QIAGEN) accordingto the manufacturer's directions. Plasmid DNA was digested with EcoRIand analyzed by agarose gel electrophoresis. Sixteen plasmid isolatescontained an EcoRI fragment of ˜800-bp, and were retained for sequencing(ABI cycle sequencer, PE Biosystems) using the vector-specific M13 andM13 reverse primers (see above).

Seqweb Gap (Genetics Computer Group) analysis indicated the optimalalignment of the sequences of the BSL3 Incyte Genomics clones and theBSL3 5′ RACE product. Seqweb Gap analysis (Genetics Computer Group) alsoindicated that the BSL3 nucleotide sequence shared less than 55%sequence identity with B7-1, B7-2, or BSL1/B7-H1 nucleotide sequences,while the BSL3 amino acid sequence shared less than 45% sequenceidentity with the B7-1, B7-2, and BSL1/B7-H1 amino acid sequences.

In addition, the BSL3 C-terminal amino acid sequence shared less than98% identity to the amino acid sequence encoded by GenBank Accession No.AK001872, over a stretch of 174 amino acids. Amino acid sequencealignments using the GCG Gap program indicated that the longest stretchof identical residues shared by BSL3 and AK001872 was 99 contiguousamino acids in length. Nucleotide sequence alignments using GCG Gapindicated that the longest stretch of identical bases shared by BSL3 andAK001872 was 239 contiguous nucleotides in length. Notably, a mouseorthologue of BSL3 was identified from the GenBank Database (AccessionNo. AF142780). The BSL3 N-terminal amino acid sequence sharedapproximately 70% sequence identity with the amino acid sequencecorresponding to mouse AF142780, over a stretch of 250 amino acids. Inaddition, it was noted that BSL3 has also been identified as PD-L2, anapparent inhibitor of T-cell proliferation (Y. Latchman et al. (2001)Nature Immunology 2:261-268).

The nucleotide and predicted amino acid sequences of BSL3 are shown inFIGS. 5A-5B. The plasmid carrying DNA encoding BSL3 (pCR2.1:BSL3) wasdeposited with the American Type Culture Collection (ATCC, 10801University Blvd., Manassas, Va. 20110-2209 USA), under ATCC DesignationNo. PTA-1986, on Jun. 6, 2000. It is noted that all ATCC depositsdescribed herein were made in accordance with the Budapest Treaty.

The BSL1, BSL2, and BSL3 sequence information is summarized in Table 5.TABLE 5 Nucleic Acid Amino Acid Sequence (NA) SEQ NA FIG (AA) SEQ AAFIG. BSL NO. Name ID NO: NO. ID NO: NO. 1 BSL1 (TNF-α) 1 1A 2 1B 1 BSL1(GM-CSF/IL-4) 3 1C 2 1B 1 BSL1-Ig 4 2A 5 2B 2 BSL2-4616811 6 and 131 3Aand 3G 7 3B (BSL2vcvc) 2 BSL2-L165-21 10 3C 11 3D (BSL2v2c2) 2BSL2-L165-35b 12 3E 13 3F (BSL2v1c2) 2 BSL2-4616811-Ig 8 4A 9 4B(BSL2vcvc-Ig) 2 BSL2-L165-35b-Ig 132 4C 133 4D (BSL2v1c2-Ig) 2BSL2-L165-21-Ig 134 4E 135 4F (BSL2v2c2-Ig) 3 BSL3-L143 14 5A 15 5B 3BSL3-L232-6-Ig 16 6A 17 6B

Example 6 Characterization of BSL3

BSL3 expression analysis: BSL3 expression patterns were determined byNorthern blot analysis of various cell types, using the BSL3 probe shownin FIG. 7C, and the procedure described for BSL1. A 2.7-kb BSL3 mRNAtranscript was detected in several cell types. In particular, highlevels of BSL3 mRNA were detected in all HMVEC stimulated with TNF-alpha(FIG. 7D). Moderate levels of BSL3 mRNA were detected in peripheralblood monocytes stimulated with PHA or GM-CSF/IL-4 (FIG. 7D). However,or BSL3 mRNA was not detected in any of the remaining cell types (FIG.7D).

In addition, multiple tissue Northern blots and expression arrays werepurchased from CLONTECH Laboratories and hybridized with P³²-labeledBSL3 probe. Briefly, a 900-bp BSL3 fragment (BSL3/KpnI+XbaI) wasisolated from clone L168-2 using KpnI and XbaI restrictionendonucleases. The fragment was run on a 1.2% agarose gel, and purifiedusing the QIAGEN Gel Extraction Kit. Approximately 30 ng ofBSL3/KpnI+XbaI was radiolabeled (6000 Ci/mmol P³²-dCTP) using the RandomPrimed DNA Labeling Kit (Roche, Indianapolis, Ind.). Unincorporatednucleotides were removed using NucTrap Probe Purification Columns(Stratagene, La Jolla, Calif.). Radiolabeled BSL3/KpnI+XbaI probe wasadded at a specific activity of 3.0×10⁶ cpm/ml of ExpressHybhybridization solution (CLONTECH) and incubated overnight at 65° C.Blots were washed with 0.1×SSC/0.1% SDS at 62° C. and exposed to filmfor 72 hr.

Northern blot analysis indicated that high levels of BSL3 transcriptwere present in spleen tissue; moderate levels of BSL3 transcript werepresent in thymus, testis, ovary, and small intestine tissues; and lowlevels of BSL3 transcript were present in heart, placenta, lung, liver,skeletal muscle, prostate, colon, lymph node, trachea, and adrenal glandtissues, and Burkitt's lymphoma RAJI cell line (FIG. 7E). Microarrayanalysis indicated that high levels of BSL3 transcript were present inspleen tissue; moderate levels of BSL3 transcript were present in lung,liver, placenta, fetal spleen, lymph node, and fetal thymus tissues; andlow levels of BSL3 transcript were present in heart, aorta, corpuscallosum, left atrium, right atrium, jejunum, thymus, fetal liver, andmammary gland tissues (FIG. 7F).

Quantitative PCR: BSL1 and BSL3 expression patterns were determined byquantitative PCR. Human Multiple Tissue cDNA (MTC™) Panels (Cat. #PT3158-1) were purchased from CLONTECH. For each PCR reaction, 5 μl ofcDNA was used. Human and murine BSL1 and BSL3 PCR primers were designedby Primer3 program (Whitehead Institute for Biomedical Research; SteveRozen, Helen J. Skaletsky (1998) Primer3) as shown in Table 6. TABLE 6SEQ ID nucleo- Primer Sequence NO: tides human BSL1 TACAAGCGAATTACTGTGAA83 459-479 forward human BSL1 GATGTGCCAGAGGTAGTTCT 84 773-793 reversehuman BSL3 AATAGAGCATGGCAGCAATG 85 419-439 forward human BSL3GGCGACCCCATAGATGATTA 86 634-654 reverse human 18s rRNACCAGTAAGTGCGGGTCAT 87  7-25 forward human 18s rRNA TTCACCTACGGAAACCTT 88196-214 reverse

SYBR Green PCR Core Reagents (Cat. # 4306736) were purchased from PEApplied Biosystem. Real-time PCR was performed on ABI Prism® 5700Sequence Detection System PE Applied Biosystem. PCR samples wereincubated at 95° C. for 15 sec, 55° C. for 20 sec, and 75° C. for 1 minfor 40 cycles. The BSL1 PCR product was 334-bp; the BSL3 PCR product was235-bp; the 18S rRNA PCR was 207-bp. Following PCR and data collection,dissociation curve studies were performed. In addition, PCR samples wereanalyzed by agarose gel electrophoresis to confirm the size of the PCRproduct.

Data processing and presentation: For each PCR reaction, a thresholdcycle number (CT) was generated as a read-out by the real-time PCRmachine. The data was processed according to the manual of ABI Prism®5700 Sequence Detection System. Briefly, the C_(T) of BSL1 and BSL3 wasnormalized to the C_(T) of 18S rRNA in the each sample. The data wasthen subtracted by the normalized C_(T) in the sample showing lowestexpression levels. For BSL1, the lowest expression levels were found intonsil tissue. For BSL3, the lowest expression levels were found inskeletal muscle. The final data was presented as the fold-increase overthe lowest expression levels.

Quantitative PCR indicated that high levels of BSL1 transcript werepresent in lymph node, spleen, lung, and placenta tissue; moderatelevels of BSL1 transcript were present in thymus and pancreas tissue;and low levels of BSL1 transcript were present in heart, brain, kidney,adult liver, skeletal muscle, bone marrow, leukocyte, fetal liver, andtonsil tissue (FIG. 7G). For BSL3, high levels of transcript werepresent in lymph node and spleen tissue; and low levels of BSL3transcript were present in thymus, lung, tonsil, heart, pancreas,placenta, kidney, bone marrow, fetal liver, and adult liver tissue (FIG.7H).

BSL3-Ig (L232-6) fusion construct: The BSL3-Ig (L232-6) construct wasmade using the following procedure. The ECD of BSL3 was amplified by PCRusing 0.01 μl pCR2.1:BSL3 (L143-4), described above; 5 μl GibcoBRL PCRbuffer; 1.5 μl of 25 mM MgCl₂; 1 μl of 10 mM dNTPs; 2.5 μl BSL3-3 primer(10 pM/μl); 2.5 μl BSL3Ig-6 primer (10 pM/μl); 1 μl CLONTECH Advantagepolymerase; and 36 μl dH₂O. The primers contained the followingsequence: BSL3-3: 5′ cc gg ggt acc ATG ATC TTC CTC CTG CTA ATG TTG 3′(SEQ ID NO:89); BSL3Ig-6: 5′ cg gaa ttc GGT CCT GGG TTC CAT CTG 3′ (SEQID NO:90). PCR samples were incubated at 94° C. for 1 min; followed by20 cycles of incubation at 94° C. for 30 sec, 59° C. for 30 sec, and 72°C. for 30 sec; followed by incubation at 72° C. for 10 min.

Following this, 38 μl of the PCR product was digested with KpnI/EcoRIand run on a 1.2% agarose/0.5×TBE gel. A band of about 650-bp wasexcised and purified with QIAGEN gel extraction kit. Ten microliters ofthe purified fragment was run on an agarose gel next to a size standard.Ten nanograms of fragment (L232) was ligated to 20 ng of KpnI/EcoRIdigested L200-1 (described herein). Five microliters of the ligationmixture was used for transformation into GibcoBRL MAX EfficiencyDH5-alpha competent bacteria, and transformants were plated onto LBplates containing 100 μg/ml ampicillin. Plates were incubated at 37° C.for 18 hr. Colonies were inoculated into 5 ml of LB broth containing 100μg/ml ampicillin and grown for 18 hr at 37° C. DNA was purified usingQIAGEN spin miniprep kit and digested with PmeI. The digested sampleswere run on an agarose gel and plasmids that contained fragments ofabout 1500-bp were sequenced. L232-6 was determined to have wild-typeBSL3 sequence. The nucleotide and predicted amino acid sequence ofBSL3-Ig (L232-6) is shown in FIGS. 6A-6B.

BSL3-Ig (L275-1) fusion construct: The BSL3-Ig (L275-1) construct wasmade using the following procedure. BSL3-Ig was PCR amplified fromL232-6 using 0.001 μL232-6; 5 μl GibcoBRL PCR buffer; 1.5 μl 25 mMMgCl₂; 1 μl 10 mM Boehringer-Mannheim dNTPs; 2.5 μl BSL3-5 primer (10pM/μl); 2.5 μl BSL1Ig-2 primer (10 pM/μl); 1 μl CLONTECH Advantagepolymerase; and 35.5 μl dH₂O. The primers contained the followingsequences: BSL3-5: 5′ cg gga ttc ATG ATC TTC CTC CTG CTA ATG TT 3′ (SEQID NO:91); and BSL1Ig-2: 5′ gc gc tct aga TCA TTT ACC CGG AGA CAG G 3′(SEQ ID NO:92). PCR samples were incubated at 94° C. for 1 min; followedby 20 cycles of incubation at 94° C. for 30 sec, 58° C. for 30 sec, and72° C. for 1.5 min; followed by incubation at 72° C. for 10 min.

Following this, 2 μl of the PCR reaction was ligated (L262) into pCR2.1using the TA cloning kit (Invitrogen). Five microliters of the ligationwas used for transformation into MAX Efficiency DH5-alpha competentcells (GibcoBRL), and transformants were plated onto LB platescontaining 100 μg/ml ampicillin and 800 μg of X-Gal. Plates wereincubated at 37° C. for 18 hr. White colonies were inoculated into 5 mlof LB broth containing 100 μg/ml ampicillin and grown at 37° C. for 18hr. Plasmid DNA was purified using QIAGEN spin miniprep kit. Plasmid DNAwas digested with BamHI/XbaI and analyzed on an agarose gel. Plasmidsthat contained an approximately 1300-bp fragment were sequenced. L262-2was determined to contain the wild-type BSL3 sequence.

L262-2 was digested with BamHI/XbaI and run on a 1.2% agarose/0.5×TBEgel. An approximately 1300-bp fragment was purified using QIAGEN gelextraction kit. Ten nanograms of the purified fragment was ligated into30 ng of BamHI/XbaI digested pD18 (related to pD16 and pD17 plasmids,described in U.S. Pat. No. 6,051,228). Five microliters of the ligationwas used for transformation into GibcoBRL MAX Efficiency DH5-alphacompetent bacteria. Transformants were plated onto LB plates containing100 μg/ml ampicillin and grown at 37° C. for 18 hr. Colonies wereinoculated into 5 ml of LB broth containing 100 μg/ml ampicillin andgrown at 37° C. for 18 hr. Plasmid DNA was purified using QIAGEN spinminiprep kit. Plasmid DNA was digested with BamHI plus XbaI or HindIII,and the digested samples were analyzed on an agarose gel. As determinedby restriction mapping, L275-1 through L275-9 contained the correctconstruction.

Purification of BSL3-Ig fusion Protein: Purification of BSL3-Ig(human-IgG1) was accomplished by one-step affinity purification.Supernatant from transiently transfected COS cells expressing BSL3-Igwas applied to a Sepharose column of immobilized protein A. The columnwas washed with PBS until the absorbance at 280 nm reached the baselinelevel. Bound protein was eluted with ImmunoPure IgG Elution Buffer(Pierce Chemical, Rockford, Ill.; Cat. # 21004). Fractions containingthe bound protein were neutralized with 1/8 v/v of 3 M sodium phosphate,pH 7. The resulting preparation was dialyzed against PBS, filtered (0.2μm). All buffers contained 0.02% w/v sodium azide.

Immunization with BSL3 polypeptide: For the initial immunization, micebetween 1 and 3 months were used (BalbC; Harlan, Indianapolis, Ind.).RIBI adjuvant was prepared as follows. In one vial, 0.5 mg MPL(monophosphoryl lipid A; RIBI Immunochemical Research, Inc., Hamilton,Mont.); 0.5 mg TDM (synthetic trehalose dicorynomycolate; RIBIImmunochemical Research, Inc.); and 40 μl squalene with 0.2% Tween®D80were mixed together. The mixture was warmed to 40-45° C. for 5-10 min,and 2 ml of BSL3 polypeptide/PBS (125 μg/ml) was added. The solution wasvortexed vigorously for several minutes. The solution was drawn into asyringe and injected immediately into the mice.

Dosages followed recommendations by RIBI Immunochemical Research, Inc.For the first injection, approximately 100 μg of BSL3 polypeptide wasresuspended in 250 μl of 1×RIBI in PBS. The mixture was injectedintraperitoneally with a 21 gauge needle. For second and laterinjections, boosts were given at least 3 weeks apart. Injections were athalf dose. At least 3 weeks following the third injection, once thetiter reached an acceptable level (see below), the animal was given afinal boost. For final injections, approximately 1 mg/ml of BSL3polypeptide was resuspended in PBS (RIBI was omitted), and the mixturewas administered intravenously via tail veins. Animals were harvested3-4 days later.

To monitor titer levels, sera samples were taken before initialimmunization (background) and 7-10 days after each immunization fortiter monitoring. Titer levels were measured by ELISA. Sera washarvested by eye-bleed or tail-bleed. Typically, 200 μl of blood wasremoved for sera testing.

Hybridoma cell lines: Hybridoma cell lines were constructed using thefollowing reagents: Iscoves Modified Dulbecco's Medium (GibcoBRL; Cat.#12440-053); fetal bovine serum (Hyclone; Cat. # A-1115L, Lot #11152564) heat inactivated for 30 min at 56° C.; L-glutamine-200 mm, 100X (GibcoBRL; Cat. # 25030-081); penicillin/streptomycin (GibcoBRL; Cat.# 15140-122); ORIGEN Hybridoma Cloning Factor (Igen International, Inc.,Gaithersburg, Md.; Cat. # IG50-0615, Lot #8077); HT Supplement 100 X(GibcoBRL; Cat. # 11067-030); HAT Supplement 100 X (GibcoBRL; Cat. #31062-037); PEG 1500, (Boehringer Mannheim; Cat. # 783 641); Red BloodCell Lysing Buffer from Lab Services (Cat. # 3KL-449); Trypan Blue; 70%ethanol; and myeloma cells (P3x) from ATCC.

The following equipment and supplies were used: laminar flow hood; CO₂incubator; inverted microscope; 37° C. water bath; centrifuge; 96-wellflat bottom tissue culture plates (Corning; Cat. # 25860-96);Serological pipettes and Integrid petri dishes (Falcon); 50 mlcentrifuge tubes (Corning; Cat. # 430921); 15 ml conical tubes (Falcon;Cat. # 2096); autoclaved scissors and forceps; multichannel pipette;wide orifice 200 μl pipette tips (Denville Scientific, Inc., Metuchen,N.J.; Cat. # P1105-CP); and sterile pipette tips (VWR, Buffalo Grove,Ill.; Cat. # 53508-794).

HAT and HT medium were made as follows: HAT Medium HT Medium IMDM 500 mlIMDM 500 ml L-Glutamine  2.5 ml L-Glutamine  2.5 ml Pen/Strep  5 mlPen/Strep  5 ml HAT Supplement  5 ml HT Supplement  5 ml Origen Hy.Clon. F. 10% Final Origen Hy. Clon. F. 10% Final

Mice were given a final boost 3-4 days prior to fusion in PBSintravenously or intraperitoneal. Myeloma cells were kept in exponentialgrowth (log phase). The mice were euthanized, and the spleen from eachmouse was aseptically harvested. The spleen was placed in a petri dishcontaining warm Iscoves solution without FBS. A spleen cell suspensionwas prepared and transferred to a centrifuge tube. HAT-sensitive myelomacells were placed in a separate 50 ml centrifuge tube. Spleen cells andmyeloma cells were centrifuged at 400×g for 5 min, and the supernatantwas aspirated. The red blood cells from the spleen were lysed with 5 mlRBC Lysing Buffer for 1 min, and the tube was filled with SF media(hybridoma serum-free media; GibcoBRL; Cat. # 12045-076). Splenocyteswere washed with 50 ml SF media, and spleen cells and myeloma cells werecentrifuged in separate centrifuge tubes at 400×g for 5 min. Spleencells and myeloma cells were counted and resuspended in 25 ml with SFmedia. Myeloma cells were added to spleen cells to give a 1:4 ratio. Themixture was centrifuged at 400×g for 10 min to form a tight pellet, andall media solution was removed by aspiration.

For the cell fusion experiments, PEG, SF media, and HAT media wereincubated at 37° C. One milliliter of 50% PEG was added to the cells for1 min (PEG was added for 30 sec and cells were stirred for 30 sec). ThePEG solution was added to the side of the tube, and the pellet wasgently stirred. With stirring, 1 ml SF media was added to the cells for1 min, and 8 ml of SF media was added to the cells for 2 min. Cells werecentrifuged at 400×g for 10 min, and the supernatant was aspirated.Cells were gently resuspended in 10 ml HAT selective media by aimingpipette directly at pellet and stirring. Additional HAT media was addedto bring cell concentration to 5×10⁵ cells/ml. Cells were aliquottedinto 96-well tissue culture plates at 5×10⁴ cells/well. After 3 days, HTmedia was added at 100 μl/well. Approximately 10 days later, clones weretested for antibody production. Positive clones were expanded to onewell of a 24-well plate. Positive clones were then re-tested, isotyped,and expanded to T25 (0.25 cm square tissue culture flask).

ELISA analysis: To test for positive clones, ELISA analysis wasperformed using the following reagents and supplies:carbonate/bicarbonate pH 9.6 (Sigma, St. Louis, Mo.; Cat. # C-3041) forcoat; Immulon 2 ELISA plates (Dynex, Chantilly, Va.; Cat. # 0110103455);10×PBS (GibcoBRL) made to 1× concentration; wash buffer comprisingTween®20 (0.05% final concentration) in 1×PBS; block buffer/samplediluent comprising wash buffer with 5% NFM non-fat milk; and chromogenmixture comprising 50% TMB (Kirkegaard & Perry Labs Gaithersburg, Md.;Cat. # 50-76-01) and 50% peroxidase (Kirkegaard & Perry Labs Cat. #50-65-00).

For ELISA, plates were coated with 75 μl/well (1 μg/ml) BSL3-Ig incarbonate/bicarbonate overnight at 4° C. Plates were washed with PBSTween®20 (using Skatron), and blocked with 300 μl block buffer for 45min at room temperature. Plates were flicked dry and incubated with 75μl/well sera diluted in blocking buffer (sera was diluted 1:50 forhighest concentration and then serially diluted by factors of three) for45 min at room temperature, and washed as before. Plates were thenincubated with 75 μl/well anti-mouse IgG in blocking buffer (1:10000dilution; HRP-labeled; Amersham Pharmacia Biotech, Piscataway, N.J.) for45 min at room temperature, and washed as before. Following this, plateswere incubated with 100 μl/well chromogen mixture, and incubated up to15 min at room temperature. The signal was quenched with 100 μl 1Nsulfuric acid, and samples were read at 450/630 nm. Using ELISA,supernatants from hybridomas were initially screened against BSL3-Igfusion protein, and then screened against Ig protein alone. Hybridomasthat produced antibodies that bound to BSL3-Ig, but not Ig, weredesignated as positive clones.

Subcloning: Positive clones from the initial fusion plate were expanded.Once growing, cells were put through two rounds of single cell cloningto ensure that they were monoclonal. Each hybridoma was plated in a96-well tissue culture plate at a concentration of 0.5 cells/well orless. Once macroscopic colonies formed, supernatants were screened byELISA. Positive clones from each hybridoma were titered by ELISA. Theclones giving rise to the strongest signals were expanded and putthrough a second round of cloning. Positive clones were again screenedby ELISA and titered. The clones giving rise to the strongest signalwere expanded and frozen back.

Example 7 Assays Using BSL Monoclonal Antibodies

FACS Analysis of Lung Epithelial Cells Using BSL1 and BSL2 MonoclonalAntibodies: A549, a lung epithelium cell line was cultured in RPM1 1640(GibcoBRL Cat. # 11875-005) plus 10% FBS (Summit, Ft Collins, Colo.;Cat. # S-100-05) and 1% penicillin-streptomycin (GibcoBRL Cat. #15140-122) at 37° C. in 5% CO₂ to 90% confluence in a T75 flask (BectonDickinson, Franklin Lakes, N.J.; Cat. # 353111). Cells were lifted withVersene (GibcoBRL Cat. # 15040-066) and washed twice in RPMI 1640. Cellswere added to a 96-well plate (Becton Dickinson Cat. # 353077; 2.5×10⁵cells per well), and centrifuged at 2000 RPM in a Beckman tabletopcentrifuge. Next, cells were resuspended in RPMI 1640 or RPMI 1640 with1 μg negative control antibody (MAb 15E10AA3; also called MAb 15E10A3,MAb 3-13E10A3, or MAb 3_(—)15) or hybridoma supernatant, and incubatedon ice for 30 min. It is noted that MAb 15E10AA3 does not recognize theBSL2 or BSL3 polypeptides, but is the identical isotype as the anti-BSL2antibodies. Cells were then washed twice in RPMI 1640, and resuspendedin goat anti-mouse anti-Fc FITC conjugated antibodies (BioSource,Camarillo, Calif.; Cat. # AMI4408) diluted 1:50 in RPMI 1640. Followingthis, cells were incubated on ice for 30 min, washed twice in RPMI 1640,resuspended in RPMI 1640, and analyzed on a Becton Dickinson FACScan.Results of FACS analysis are shown for anti-BSL1 MAb 32F9A7 (FIG. 9A)and anti-BSL2-4616811 MAbs (FIG. 9B). It is noted that all MAbs bound tothe A549 cells. It is also noted that anti-BSL2 MAb 1F7G2, anti-BSL2 MAb2B10D7, anti-BSL2 MAb 3E6D3, anti-BSL2 MAb 4C2C6, and anti-BSL2 MAb5D7E2 are also termed anti-BSL2-1 MAb, anti-BSL2-2 MAb, anti-BSL2-3 MAb,anti-BLS24 MAb, and anti-BSL2-5 MAb, respectively, as described herein.

FACS Analysis of Various Cell Types Using BSL3 Monoclonal Antibodies: Todetermine whether BSL3 polypeptide was expressed on the surface ofvarious cell types, cells were grown in media as shown in Table 7,below. HUT 78 was supplemented with 20% FBS (Summit Biotechnology, FtCollins, Colo.; Cat. # S-100-05). All other cell lines were supplementedwith 10% Summit FBS. In addition, all cell lines were supplemented with1% penicillin/streptomycin (GibcoBRL; Cat. # 15140-122). All cell lineswere grown at 37° C. in 5% CO₂. TABLE 7 CELL LINE ORIGIN MEDIA HL60 premyeloid RPMI 1640 THP1 monocytic RPMI 1640 A549 lung epithelium RPMI1640 H292 lung epithelium RPMI 1640 PM LCL B lineage RPMI 1640 PL LCL Blineage RPMI 1640 CE LCL B lineage RPMI 1640 RAMOS B lineage RPMI 1640CEM T lineage RPMI 1640 HUT 78 T lineage IMDM¹ Jurkat T lineage RPMI1640²¹IMDM: GibcoBRL; Cat. # 12440-053²RPMI 1640: GibcoBRL; Cat. # 11875-005

Cells were grown to about 5×10⁵ cells/min. Cells were washed twice inserum free RPMI 1640 and resuspended to give a final concentration of2.5×10⁶ cells/ml. Anti-BSL3 MAb 1A4A1 antibodies or isotype control MAb15E10A3 antibodies (also called MAb 15E10AA3, MAb 3-15E10A3, and MAb3_(—)15) were added to 5 μg/ml and incubated at 4° C. for 30 min. Cellswere washed twice in serum free RPMI 1640 and resuspended in serum freeRPMI 1640 plus 2% goat anti-mouse IgG conjugated to FITC (BioSource,Camarillo, Calif.; Cat. # AMI4408). Cells were incubated 30 min at 4° C.Cells were washed twice in serum free RPMI 1640 and analyzed on a BectonDickinson FACScan (Becton Dickinson, Franklin Lakes, N.J.). BSL3polypeptide was expressed on A549 (lung epithelium), H292 (lungepithelium), PM LCL (B lineage), PL LCL (B lineage), CE LCL (B lineage),and HUT78 (T lineage) cells (FIG. 9C).

FACS Analysis of Human Umbilical Vein Endothelial Cells using BSL3Monoclonal Antibodies: Anti-BSL3 monoclonal antibodies were used tomeasure BSL3 polypeptide levels on human umbilical vein endothelialcells with or without TNF-alpha stimulation. Human umbilical veinendothelial cells (HUVEC) were grown to confluence at 37° C. with 5% CO₂in EGM media (Clonetics, Walkersville, Md.; Cat. # CC4176). TNF-alphawas omitted or added to 10 ng/ml for 24 hr. Cells were lifted withVersene (GibcoBRL; Cat. # 15040-066) and prepared for flow cytometryusing anti-BSL3 MAb 1A4A1 antibodies as described above. As a control,flow cytometry was performed without antibodies. The results indicatedthat BSL3 polypeptide levels increased on TNF-alpha stimulated HUVEC(FIG. 9D). This increase was not observed in unstimulated cells (FIG.9D).

FACS Analysis of Human Peripheral Blood Monocytes using BSL3 MonoclonalAntibodies: Anti-BSL3 monoclonal antibodies were used to measure BSL3polypeptide levels on human peripheral blood monocytes with or withoutGM-CSF IL-4 or PHA stimulation. Peripheral blood monocytes (PBMC) werepurified as follows. Blood samples were aliquotted equally among twelve50 ml conical tubes. One volume of elutriation buffer (at roomtemperature) was added to each of the samples. Samples were underlayedwith 10 ml LSM (lymphocyte separation mixture) ficoll solution, andcentrifuged at 1800 rpm for 25 min. The upper layer of reddish materialwas removed, and the LSM layer was transferred to a new tube (6 tubesper donor). Most of the PBMC were observed on top of the LSM layer.Elutriation buffer (50 ml) was added to each tube, and the mixture wascentrifuged at 1800 rpm for 8 min. The supernatant was aspirated, andthe PBMC were resuspended in 15 ml elutriation buffer. The mixture wastransferred into 2 new 50 ml conical tubes. (45 ml total volume pertube), and centrifuged at 1000 rpm for 8 min. The supernatant wasaspirated, and this process was repeated two more times.

PBMC were resuspended in flasks containing RPMI 1640 with 2% FBS, andincubated at 37° C. in 5% CO₂ for 1 hr. Flasks were rocked once every 20min. Flasks were washed gently (twice) with media to remove T-cells andB cells. Flasks were then washed vigorously with RPMI 1640 plus 10% FBSand 1% penicillin/streptomycin to obtain monocytes. PBMC were washedtwice in RPMI 1640 with 10% FBS and 1% penicillin/streptomycin. PBMCwere resuspended to 5×10⁶ cells/ml, and transferred to flasks. PBMC wereincubated at 37° C. in 5% CO₂ without stimulation for 4 days. Inparallel experiments, PBMC were incubated with GM-CSF (15 ng/ml) andIL-4 (75 ng/ml) for 4 days, or PBMC were incubated with PHA (1 μg/ml)for 4 days. Flasks were washed vigorously with RPMI 1640 to remove themonocytes. PBMC were washed twice in RPMI 1640 examined by flowcytometry as described for the various cell lines, above. The resultsindicated that BSL3 polypeptide levels increased on GM-CSF IL4 or PHAstimulated cells (FIGS. 9E-9F). This increase was not observed inunstimulated cells (FIGS. 9E-9F).

Peripheral Blood T Cell Costimulation: 96-well plates (Becton DickinsonCat. # 353072) were coated with the indicated amount of anti-CD3 MAbG19.4 (described herein) in PBS (GibcoBRL Cat. # 14190-144). Plates wereincubated at 4° C. for 16 hr. Plates were washed twice in PBS. Thefollowing proteins were added: BSL3-Ig (15 μg/ml) or Chi L6 (10 μg/ml)in PBS. Chi L6 is a protein fragment that comprises the Fc portion ofhuman IgG, and is identical to the Fc portion used in the BSL-Ig fusionproteins, described above. Different concentrations of protein were usedto give equivalent molarity. Plates were incubated at 37° C. for 4 hr.Plates were washed twice in PBS. Peripheral blood T-cells were purifiedas described, above. Cells were added (5×10⁴ cells per well) in RPMIwith 10% human serum (Sigma; Cat. # H4522) and 1%penicillin/streptomycin. Cells were incubated at 37° C. in 5% CO₂ for 72hr. During the last 8 hr, cells were incubated with an additional 50 μlof media containing 50 μCi/ml ³H-thymidine (NEN; Cat. # NET-027). Cellswere harvested on a Brandel cell harvester (Brandel, Gaithersburg, Md.)using Packard GF/C plates (Packard, Meriden, Conn.; Cat. # 6005174), andthe plates were air-dried overnight. After this, 50 μl Microscint 20(Packard; Cat. # 6013621) was added, and the radiolabel was counted on aPackard Topcount NXT.

Blockade of Peripheral Blood T Cell Costimulation Using BSL2 and BSL3Monoclonal Antibodies: 96-well plates (Becton Dickinson Cat. # 353072)were coated with 20 μg/ml anti-CD3 MAb G19.4 (described herein) in PBS(GibcoBRL Cat. # 14190-144). Plates were incubated at 4° C. for 16 hr.Plates were washed twice in PBS. The following proteins were added:BSL3-Ig (15 μg/ml) or L6-Ig (10 μg/ml) in PBS. Different concentrationsof protein were used to give equivalent molarity. Plates were incubatedat 37° C. for 4 hr. Plates were washed twice in PBS. Peripheral bloodT-cells were purified as described, above. Cells were added (5×10⁴cells/well) in RPMI with 10% human serum (Sigma; Cat. # H4522) and 1%GibcoBRL penicillin/streptomycin. Purified anti-BSL3 MAbs or controlisotype MAbs were added to a final concentration of 20 μg/ml. To assayco-stimulation, monoclonal antibodies were omitted. Plates wereincubated at 37° C. in 5% CO₂ for 72 hr. During the last 8 hr, cellswere incubated in an additional 50 μl of media with 50 μCi/ml³H-thymidine (NEN; Cat. # NET-027). The cells were harvested on aBrandel cell harvester using Packard GF/C plates (Cat. # 6005174), andthe plates were-air-dried overnight. Following this, 50 μl Microscint 20(Packard Cat. # 6013621) was added, and the radiolabel was counted on aPackard Topcount NXT.

The results indicated that BSL3-Ig fusion protein acted as aco-stimulatory molecule for peripheral blood T-cells incubated withanti-CD3 MAb G19.4 (FIGS. 10A-10D). This was confirmed with separateperipheral blood T-cells donors: donor 078 (FIG. 10A) and donor 124(FIG. 10B). The results further indicated that anti-BSL3 MAbs blockedthe co-stimulatory effect of the BSL3-Ig fusion protein (FIGS. 10C-10D).This was confirmed with separate peripheral blood T-cells donors: donor010 (FIG. 10C) and donor 127 (FIG. 10D).

Peripheral Blood T Cell Assays: Peripheral blood T-cell assays wereperformed to determine the immunomodulatory properties ofBSL2-4616811-Ig (BSL2vcvc-Ig), monclonal antibodies directed toBSL2-4616811, and BSL2-L165-35b-Ig (BSL2v1c2-Ig).

1. For the first set of experiments, 100 μl of the indicatedconcentration (2, 1, 0.5, 0.25, 0.13, or 0 μg/ml) of anti-CD3 MAb G19.4(described herein) was added in triplicate to a Costar plate (CorningInc., Corning N.Y.; Cat. # 3595) in Gibco DPBS (Invitrogen Corp., GrandIsland, N.Y.). The plate was incubated at 4° C. for 16 hr. The plate waswashed two times in DPBS. Following this, 100 μl of 30 μg/mlBSL2-4616811-Ig (BSL2vcvc-Ig) or 10 μg/ml ChiL6 fusion protein was addedper well in triplicate and incubated at 37° C. for 4 hr. The plate waswashed two times in DPBS. Then, 50,000 E-rosetted peripheral bloodT-cells in 200 μl Gibco RPMI 1640 (Cat. # 11875-085) plus 1/100 volumeGibco penicillin-streptomycin (Cat. # 15140-122) plus 10% human serum(Sigma, St. Louis, Mo.; Cat. # H4522) were added per well. The plate wasincubated at 37° C. in 5% CO₂ for 2 days.

Following this, 50 μl of the same media and 1/50 volume of Perkin-Elmer³H-thymidine (Perkin-Elmer Life Sciences Inc., Boston, Mass.; Cat. #NET-027) was added per well. The plate was incubated at 37° C. in 5% CO₂for 16 hr. The plate was harvested on a Brandel harvester model CH-600using a Packard plate (Packard, Meriden Conn.; Cat. # 6005174). Then, 40μl Packard Microscint 20 (Cat. # 6013621) was added per well. The platewas counted on a Packard TopCount NXT. Data was analyzed using MicrosoftExcel 97 (Microsoft, Redmond, Wash.). Four independent experiments wereperformed, each with cells isolated from two different donors.Representative results obtained with cells isolated from donor 100 areshown in FIG. 11A.

2. The peripheral blood T-cells assay was performed as described in (1),except that a constant concentration (250 ng/ml) of anti-CD3 MAb G19.4was used, and decreasing concentrations of BSL2-4616811-Ig (BSL2vcvc-Ig;90, 30, 10, 3.3, 1.1, or 0 μg/ml) and ChiL6 (30, 10, 3.3, 1.1, 0.37, 0μg/ml) fusion proteins were used. Two independent experiments wereperformed, each with cells isolated from two different donors.Representative results obtained with cells isolated from donor 82 areshown in FIG. 11B.

3. The peripheral blood T-cells assay was performed as described in (1),except that 40 ng/ml anti-CD3 MAb G19.4 was used and the plate wasincubated 37° C. for 4 hr. The plate was washed twice in DPBS. Then, adecreasing concentration (100 μl of 2, 1, 0.5, 0.25, 0.13, or 0 μg/ml)of anti-CD28 MAb 9.3 (described herein) was added to each well. Theplate was incubated at 4° C. for 16 hr. The plate was washed twice inDPBS and 100 μl of 90 μg/ml BSL2-4616811-Ig (BSL2vcvc-Ig) or 30 μg/mlChiL6 was added per well in triplicate. Four independent experimentswere performed, each with cells from two different donors.Representative results obtained with cells isolated from donor 50 areshow in FIG. 11C.

4. The peripheral blood T-cells assay was performed as described in (1),except that a constant concentration (200 ng/ml) of anti-CD3 MAb G19.4was used, and a decreasing concentration of BSL2-4616811-Ig(BSL2vcvc-Ig; 120, 60, 30, 15, 7.5, or 0 μg/ml), BSL2-L165-35b-Ig(BSL2v1c2-Ig; 80, 40, 20, 10, 5, or 0 μg/ml) or ChiL6 (80, 40, 20, 10,5, or 0 μg/ml) was added. Two independent experiments were performed,each with cells isolated from two different donors. Representativeresults obtained with cells isolated from donor 44 are shown in FIG.11D.

5. The peripheral blood T-cells assay was performed as described in (1),except that the plate was initially coated with 250 ng/ml anti-CD3 MAbG19.4. Following washes, the plate was coated with 30 μg/mlBSL2-4616811-Ig (BSL2vcvc-Ig). Following additional washes, anti-BSL2-1MAb, anti-BSL2-5 MAb, or non-specific 3_(—)15 MAb (also called MAb3-15E10A3, MAb 15E10A3, and MAb 15E10AA3) was added at decreasingconcentrations (54, 18, 9, 2.2, 0.74, or 0 μg/ml) in 100 μl media.T-cells were added in 100 μl media. Two independent experiments wereperformed, each with cells isolated from two different donors.Representative results obtained with cells isolated from donor 117 areshown in FIG. 11E.

6. The peripheral blood T-cells assay was performed as described in (1),except that the plates were coated with a constant concentration (200ng/ml) of anti-CD3 MAb G19.4. Following washes, the plate was coatedwith 30 μg/ml BSL2vcvc-Ig isolated from CHO cells (see above). Followingadditional washes, either anti-BSL2-1 MAb, anti-BSL2-2 MAb, anti-BSL2-3MAb, anti-BSL2-4 MAb, or non-specific MAb 3_(—)15 was added atdecreasing concentrations (20, 10, 5, 2.5, 1.25, or 0 μg/ml) in 100 μlmedia. T-cells were added in 100 μl media. The final concentrations ofthe antibodies were 10, 5, 2.5, 1.25, 0.63, 0 μg/ml. Two independentexperiments were performed, each with cells isolated from two differentdonors. Representative results obtained with cells isolated from donor10 are shown in FIG. 11F.

7. The peripheral blood T-cells assay was performed as described in (1),except that the plates were coated with a constant concentration (200ng/ml) of anti-CD3 MAb G19.4. Following washes, the plate was coatedwith 30 μg/ml ChiL6 fusion protein. Following additional washes, eitheranti-BSL2-1 MAb or non-specific 3_(—)15 MAb was added at decreasingconcentrations (40, 20, 10, 5, 2.5, or 0 μg/ml), or no antibody wasadded, in 50 μl media. T-cells were added in 150 μl media. The finalconcentrations of the antibodies were 10, 5, 2.5, 1.25, 0.63, or 0μg/ml. One experiment was performed with cells isolated from twodifferent donors. Representative results obtained with cells isolatedfrom donor 12 are shown in FIG. 11G.

8. The peripheral blood T-cells assay was performed as described in (1),except that the plates were coated with a constant concentration of 200ng/ml anti-CD3 MAb G19.4. Following washes, the plate was coated with 30μg/ml BSL3-L165-35b-Ig (BSL2v1c2-Ig). Following additional washes,anti-BSL2-1 MAb or non-specific 3_(—)15 MAb was added at decreasingconcentrations (20, 10, 5, 2.5, 1.25, or 0 μg/ml), or no antibody wasadded, in 100 μl media. T-cells were added in 100 μl media. The finalconcentration of the antibodies was 10, 5, 2.5, 1.25, 0.63 or 0 μg/ml.One experiment was performed with cells isolated from two differentdonors. Representative results obtained with cells isolated from donor12 are shown in FIG. 11H.

9. The peripheral blood T-cells assay was performed as described in (1),except that the plates were coated with a constant concentration (200ng/ml) of anti-CD3 MAb G19.4. Following washes, the plate was coatedwith 30 μg/ml BSL2-4616811-Ig (BSL2vcvc-Ig) isolated from CHO cells (seeabove). Following additional washes, the plate was coated withdecreasing concentrations (10, 5, 2.5, 1.25, 0.63, or 0 μg/ml) ofanti-BSL2-1 MAb or non-specific 3_(—)15 MAb, or no antibody was added.Following more washes, T-cells were added in 200 μl media. Oneexperiment was performed with cells isolated from two different donors.Representative results obtained from cells isolated from donor 12 areshown in FIG. 11I.

10. The peripheral blood T-cells assay was performed as described in(1), except that the plates were coated with a constant concentration(200 ng/ml) of anti-CD3 MAb G19.4. Following washes, the plate wascoated with decreasing concentrations (10, 5, 2.5, 1.25, 0.63, or 0μg/ml) of anti-BSL2-1 MAb or non-specific 3_(—)15 MAb, or no antibodywas added. Following additional washes, the plate was coated with 30μg/ml BSL2-4616811-Ig (BS2vcvc-Ig) isolated from CHO cells (see above).Following more washes, T-cells were added in 200 μl media. Oneexperiment was performed with cells isolated from two different donors.Representative results obtained from cells isolated from donor 12 areshown in FIG. 11J.

The results of these assays are summarized as follows. In theseexperiments, BSL2-4616811-Ig (BSL2vcvc-Ig) fusion protein acted as apotent inhibitor of T-cell proliferation, even at relatively highconcentrations of anti-CD3 MAb G19.4 (FIG. 11A). The optimal inhibitoryconcentration of BSL2-4616811-Ig in a T-cell proliferation assay wasapproximately 90 μg/ml (FIG. 11B). Moreover, BSL2-4616811-Ig-mediatedinhibition of T-cell proliferation appears to be dominant over T-cellstimulation with anti-CD28 MAb 9.3 (FIG. 11C). In contrast,BSL2-L-165-35b-Ig (BSL2v1c2-Ig) appears to have no effect on T-cellproliferation (FIG. 11D).

Surprisingly, all five anti-BSL2 monoclonal antibodies (used as solublereagents) also have a potent inhibitory effect on T-cell proliferation(FIGS. 11E-11F). The inhibitory effect of the BSL2 monoclonal antibodiesrequires the presence of the BSL2-4616811-Ig (BSL2vcvc-Ig) fusionprotein (compare FIG. 11E to FIGS. 11G-11H). In addition, the BSL2monoclonal antibody anti-BSL2-1 is more effective at inhibition whensoluble, than when bound to the plate, or when bound to the plate in thepresence of BSL2-4616811-Ig (BSL2vcvc-Ig; compare FIG. 11E to FIGS.11I-11J).

From these experiments, it is clear that the BSL2-4616811-Ig(BSL2vcvc-Ig) fusion protein inhibits T-cell proliferation. Moreover, itappears that the BSL2-4616811-Ig fusion protein acts through a pathwaythat is dominant to the CD28 stimulatory pathway. Interestingly, BSL2monoclonal antibodies act synergistically with the BSL2-4616811-Igfusion protein in inhibiting T-cell proliferation. While not wishing tobe bound by theory, the mechanism of this synergy may involve thesignaling of anti-BSL2 (BSL2vcvc) MAbs through BSL2 present on T-cells,and the formation of a complex on T-cells that contains anti-BSL2 MAbsbound to endogenous BSL2 (BSL2vcvc) bound to endogenous BSL2 (BSL2vcvc)ligand, and plate-bound BSL2-Ig (BSL2vcvc-Ig) bound to endogenous BSL2(BSL2vcvc) ligand. However, other mechanisms are also possible.

Mixed Lymphocyte Reactions: In the mixed lymphocyte reactions (MLR),100,000 E-rosetted peripheral blood T-cells from donor 124 were mixedwith 100,000 elutriated peripheral blood monocytes (isolated asdescribed above) from donor 051, and BSL2-4616811-Ig, CTLA-4-Ig, orChiL6 were added. Final concentrations were 90, 30, 10, 3.3, 1.1, or0.37 μg/ml for BSL2-4616811-Ig (BSL2vcvc-Ig), 60, 20, 6.6, 2.2, 0.73,0.24 μg/ml for CTLA-4-Ig, or 30, 10, 3.3, 1.1, 0.36, 0.12 μg/ml forChiL6. The final volume was 200 μl. A Falcon plate (Becton Dickinson,Franklin Lakes N.J.; Cat. # 35-3077) was used. Media was made asdescribed above. The plate was incubated 4 days at 37° C. in 5% CO₂. Theplate was labeled, harvested, counted and the data analyzed as indicatedabove. Results are shown in FIG. 12A.

In a second set of experiments, the MLR was performed as described,except that final concentrations were 90, 45, 22.5, 11.25, 5.625, or 0μg/ml for BSL2-4616811-Ig (BSL2vcvc-Ig), 60, 30, 15, 7.5, 3.75, or 0μg/ml for BSL2-L165-35b-Ig (BSL2v1c2-Ig), or 30, 15, 7.5, 3.75, 1.875,or 0 μg/ml) ChiL6. Results are shown in FIG. 12B. The results depictedin FIGS. 12A-12B support the results shown in FIGS. 11C-11D, describedabove. In particular, the experiments show that BSL2-4616811-Ig(BSL2vcvc-Ig)-mediated inhibition of T-cell proliferation appears to bedominant over T-cell stimulation through CD28 (FIG. 12A), and thatBSL2-L165-35b-Ig (BSL2v1c2-Ig) appears to have no effect on T-cellproliferation (FIG. 12B).

Binding Comparison of Anti-BSL2 Monoclonal Antibodies to BSL2-4616811-Ig(BSL2vcvc-Ig) and BSL2-L165-35b-Ig (BSL2v1c2-Ig). Plates were coatedwith 1 μg/ml of BSL2-4616811-Ig (BSL2vcvc-Ig) or BSL2-L165-35b-Ig(BSL2v1c2-Ig) at 50 μl/well and incubated at 4° C. overnight. Wells wereaspirated and plates were blocked with 200 μl of 1% milk blockingsolution (KPL; Cat. # 50-82-00). Plates were incubated at roomtemperature for 60 min. Plates were then washed with PBS containing0.05% Tween®20 using four washes with 300 μl/well, and 2 sec betweeneach wash (Program BMR1). Anti-BSL2-1 MAb, anti-BSL2-2 MAb, anti-BSL2-3MAb, anti-BSL2-4 MAb, anti-BSL2-5, and negative control antibody 3_(—)15MAb were added (10 μg/ml; 50 μl/well) and plates were incubated at roomtemperature for 60 min. Plates were washed as described, and donkeyanti-mouse IgG (H+L) HRP-conjugated secondary antibody (JacksonImmunoResearch Laboratories, Inc., West Grove, Pa.) was added. Plateswere incubated for 60 min at room temperature, and washed as before.Following this, 50 μl/well Develop Solution (DAKO Corp., Carprinteria,Calif., Cat. # S1599) was added. Plates were incubated at roomtemperature for about 20 min. Stop Solution was then added (2.0 Nsulfuric acid; 50 μl/well). Plates were read for absorbance at 405 nm.The results shown in FIG. 13 indicate that there was no significantdifference between the binding of the anti-BSL2 monoclonal antibodies toBSL2-4616811-Ig (BSL2vcvc-Ig) or BSL2-L165-35b-Ig (BSL2v1c2-Ig).

Example 8 Identification and Cloning of VH and VL Domains of AntibodiesDirected Against the BSL1, BSL2, or BSL3 Polypeptide

V_(H) and V_(L) domains may be identified and cloned from cell linesexpressing an antibody directed against a BSL1 (e.g., SEQ ID NO:2), BSL2(e.g., SEQ ID NO:7, SEQ ID NO:11, or SEQ ID NO:13), or BSL3 (e.g., SEQID NO:15) epitope by performing PCR with V_(H) and V_(L) specificprimers on cDNA template made from the antibody expressing cell lines.Briefly, RNA is isolated from the cell lines and used as a template forRT-PCR designed to amplify the V_(H) and V_(L) domains of the antibodiesexpressed by the EBV cell lines. Cells may be lysed using the TRIzolreagent (Life Technologies, Rockville, Md.) and extracted with one-fifthvolume of chloroform. After addition of chloroform, the solution isallowed to incubate at room temperature for 10 min, and then centrifugedat 14,000 rpm for 15 min at 4° C. in a tabletop centrifuge. Thesupernatant is collected and RNA is precipitated using an equal volumeof isopropanol. Precipitated RNA is pelleted by centrifuging at 14,000rpm for 15 min at 4° C. in a tabletop centrifuge.

Following centrifugation, the supernatant is discarded and the pellet iswashed with 75% ethanol. Following the wash step, the RNA is centrifugedagain at 800 rpm for 5 min at 4° C. The supernatant is discarded and thepellet allowed to air dry. RNA is the dissolved in DEPC water and heatedto 60° C. for 10 min. Quantities of RNA can be determined using opticaldensity measurements. cDNA may be synthesized according to methodswell-known in the art and/or described herein from 1.5 to 2.5 microgramsof RNA using reverse transciptase and random hexamer primers. cDNA isthen used as a template for PCR amplification of V_(H) and V_(L)domains. Primers used to amplify V_(H) and V_(L) genes are shown inTables 8 and 9, below. TABLE 8 Primer Sequences Used to Amplify V_(L)domains SEQ Primer name Primer Sequence ID NO: Hu Vkappa1 - 5′GACATCCAGATGACCCAGTCTCC 95 Hu Vkappa2a - 5′ GATGTTGTGATGACTCAGTCTCC 96Hu Vkappa2b - 5′ GATATTGTGATGACTCAGTCTCC 97 Hu Vkappa3 - 5′GAAATTGTGTTGACGCAGTCTCC 98 Hu Vkappa4 - 5′ GACATCGTGATGACCCAGTCTCC 99 HuVkappa5 - 5′ GAAACGACACTCACGCAGTCTCC 100 Hu Vkappa6 - 5′GAAATTGTGCTGACTCAGTCTCC 101 Hu Vlambda1 - 5′ CAGTCTGTGTTGACGCAGCCGCC 102Hu Vlambda2 - 5′ CAGTCTGCCCTGACTCAGCCTGC 103 Hu Vlambda3 - 5′TCCTATGTGCTGACTCAGCCACC 104 Hu Vlambda3b -5′ TCTTCTGAGCTGACTCAGGACCC 105Hu Vlambda4 - 5′ CACGTTATACTGACTCAACCGCC 106 Hu Vlambda5 - 5′CAGGCTGTGCTCACTCAGCCGTC 107 Hu Vlambda6 - 5′ AATTTTATGCTGACTCAGCCCCA 108Hu Jkappa1 - 3′ ACGTTTGATTTCCACCTTGGTCCC 109 Hu Jkappa2 - 3′ACGTTTGATCTCCAGCTTGGTCCC 110 Hu Jkappa3 - 3′ ACGTTTGATATCCACTTTGGTCCC111 Hu Jkappa4 - 3′ ACGTTTGATCTCCACCTTGGTCCC 112 Hu Jkappa5 - 3′ACGTTTAATCTCCAGTCGTGTCCC 113 Hu Vlambda1 - 3′ CAGTCTGTGTTGACGCAGCCGCC114 Hu Vlambda2 - 3′ CAGTCTGCCCTGACTCAGCCTGC 115 Hu Vlambda3 - 3′TCCTATGTGCTGACTCAGCCACC 116 Hu Vlambda3b - 3′ TCTTCTGAGCTGACTCAGGACCC117 Hu Vlambda4 - 3′ CACGTTATACTGACTCAACCGCC 118 Hu Vlambda5 - 3′CAGGCTGTGCTCACTCAGCCGTC 119 Hu Vlambda6 - 3′ AATTTTATGCTGACTCAGCCCCA 120

TABLE 9 Primer Sequences Used to Amplify V_(H) domains Primer namePrimer Sequence SEQ ID NO: Hu VH1 - 5′ CAGGTGCAGCTGGTGCAGTCTGG 121 HuVH2 - 5′ CAGGTCAACTTAAGGGAGTCTGG 122 Hu VH3 - 5′ GAGGTGCAGCTGGTGGAGTCTGG123 Hu VH4 - 5′ CAGGTGCAGCTGCAGGAGTCGGG 124 Hu VH5 - 5′GAGGTGCAGCTGTTGCAGTCTGC 125 Hu VH6 - 5′ CAGGTACAGCTGCAGCAGTCAGG 126 HuJH1 - 5′ TGAGGAGACGGTGACCAGGGTGCC 127 Hu JH3 - 5′TGAAGAGACGGTGACCATTGTCCC 128 Hu JH4 - 5′ TGAGGAGACGGTGACCAGGGTTCC 129 HuJH6 - 5′ TGAGGAGACGGTGACCGTGGTCCC 130

Typically a PCR reaction makes use of a single 5′ primer and a single 3′primer. At times, when the amount of available RNA template is limiting,or for greater efficiency, groups of 5′ and/or 3′ primers may be used.For example, all five VH-5′ primers and all JH-3′ primers may be used ina single PCR reaction. The PCR reaction is carried out in a 50 μl volumecontaining 1×PCR buffer, 2 mM each dNTP, 0.7 U High Fidelity Taqpolymerase, 5′ primer mix, 3′ primer mix, and 7.5 μl cDNA. The 5′ and 3′primer mix of both V_(H) and V_(L) can be made by pooling together 22pmole and 28 pmole, respectively, of each of the individual primers. PCRconditions include incubation at 96° C. for 5 min; followed by 25 cyclesof incubation at 94° C. for 1 min, 50° C. for 1 min, and 72° C. for 1min; followed by an extension cycle of 72° C. for 10 min. After thereaction has been completed, sample tubes may be stored at 4° C.

PCR samples are then electrophoresed on a 1.3% agarose gel. DNA bands ofthe expected sizes (506-bp for V_(H) domains, and 344-bp for V_(L)domains) can be cut out of the gel and purified using methods well knownin the art and/or described herein. Purified PCR products can be ligatedinto a PCR cloning vector (TA vector from Invitrogen Inc., Carlsbad,Calif.). Individual cloned PCR products can be isolated aftertransformation into E. coli and blue/white color selection. Cloned PCRproducts may then be sequenced using methods commonly known in the artand/or described herein.

The PCR bands containing the V_(H) domain and the V_(L) domains can alsobe used to create full-length Ig expression vectors. V_(H) and V_(L)domains can be cloned into vectors containing the nucleotide sequencesof a heavy (e.g., human IgG1 or human IgG4) or light chain (human kappaor human lambda) constant regions such that a complete heavy or lightchain molecule could be expressed from these vectors when transfectedinto an appropriate host cell. Further, when cloned heavy and lightchains are both expressed in one cell line (from either one or twovectors), they can assemble into a complete functional antibody moleculethat is secreted into the cell culture medium. Methods usingpolynucleotides encoding V_(H) and V_(L) antibody domain to generateexpression vectors that encode complete antibody molecules are wellknown within the art.

As various changes can be made in the above compositions and methodswithout departing from the scope and spirit of the invention, it isintended that all subject matter contained in the above description,shown in the accompanying drawings, or defined in the appended claims beinterpreted as illustrative, and not in a limiting sense.

The contents of all patents, patent applications, published articles,books, reference manuals, texts and abstracts cited herein are herebyincorporated by reference in their entirety to more fully describe thestate of the art to which the present invention pertains.

1-13. (canceled)
 14. An isolated polynucleotide sequence comprising anucleic acid sequence selected from the group consisting of: (a) anisolated polynucleotide sequence comprising polynucleotides encodingamino acids 1 to 480 of SEQ ID NO:133; (b) an isolated polynucleotidesequence comprising polynucleotides encoding amino acids 2 to 480 of SEQID NO:133; and (c) an isolated polynucleotide sequence comprisingpolynucleotides encoding amino acids 29 to 480 of SEQ ID NO:133.
 15. Theisolated polynucleotide sequence of claim 14, wherein saidpolynucleotide is (a).
 16. The isolated polynucleotide sequence of claim15, wherein said polynucleotide comprises nucleotides 1 to 1440 of SEQID NO:132.
 17. The isolated polypeptide of claim 14, wherein saidpolynucleotide is (b).
 18. The isolated polynucleotide sequence of claim17, wherein said polynucleotide comprises nucleotides 4 to 1440 of SEQID NO:132.
 19. The isolated polypeptide of claim 14, wherein saidpolynucleotide is (c).
 20. The isolated polynucleotide sequence of claim19, wherein said polynucleotide comprises nucleotides 85 to 1440 of SEQID NO:132.
 21. An isolated polynucleotide sequence comprising the cDNAclone contained in plasmid BSL2v1c2Ig in ATCC Deposit No. PTA-4056. 22.A recombinant vector comprising the isolated nucleic acid molecule ofclaim
 14. 23. A recombinant host cell comprising the vector sequence ofclaim
 22. 24. A method of making an isolated polypeptide comprising: (a)culturing the recombinant host cell of claim 23 under conditions suchthat said polypeptide is expressed; and (b) recovering said polypeptide.25. The isolated polynucleotide of claim 14 wherein said nucleic acidsequence further comprises a heterologous nucleic acid sequence.
 26. Theisolated polynucleotide of claim 25 wherein said heterologous nucleicacid sequence encodes a heterologous polypeptide.